Elevated synthesis of an axonally transported protein correlates with axon outgrowth in normal and injured pyramidal tracts

Axons of the adult mammalian CNS typically fail to regenerate after injury. Among the hypotheses to account for this failure is the proposition that certain axonal proteins necessary for axon growth are expressed in much greater abundance in developing than in mature neurons, and that these proteins are not reinduced after injury to mature axons (Skene and Willard, 1981b). In the present experiments, we have found that hamster pyramidal tract neurons synthesize an acidic, 43K protein that is transported into growing axons during the first 2 weeks of postnatal development, and then declines at least an order of magnitude by the fourth postnatal week. The decline in synthesis of the 43K protein coincides with the cessation of pyramidal tract axon elongation. This protein resembles a "growth-associated protein," GAP-43, which is induced during regeneration of CNS axons in lower vertebrates. The 43K protein in hamster pyramidal tract neurons is not reinduced after axotomy in adult animals, which correlates with the failure of the injured axons to regenerate. Injury to neonatal pyramidal tract axons does not reverse or delay the decline in 43K protein synthesis. This is consistent with previous findings (Kalil and Reh, 1982) that pyramidal tract axons regrow for only a brief period after neonatal injury. Taken together, these results lend support to the hypothesis that synthesis of GAP-43 is important for axon growth in development and regeneration.     

Phosphorylation-dependent epitopes on neurofilament proteins and neurofilament densities differ in axons in the corticospinal and primary sensory dorsal column tracts in the rat spinal cord.

The highest molecular weight neurofilament protein (NF-H) is multiply phosphorylated at epitopes which can be distinguished by specific monoclonal antibodies on Western blots. Eight characterized antibodies were used in immunocytochemistry to examine the tissue distributions of phosphorylated variants of NF-H in axons of the adult rat spinal cord. The most striking difference in staining was found between axons in the cuneate tract and those in the neighboring dorsal corticospinal tract. Axons in the cuneate tract reacted intensely with antibodies to phosphorylated epitopes of NF-H and poorly with antibodies to dephosphorylated epitopes of NF-H, whereas the reverse was the case for the axons of the dorsal corticospinal tract. These differences showed that systematic variations in the phosphorylation of NF-H in long-tract axons in the central nervous system occur as a function of cell type. When the cytoskeletons of these axons were compared by electron microscopy, the neurofilaments of the cuneate fibers were seen to be more abundant and formed a latticework, more compactly organized than the neurofilaments of the dorsal corticospinal axons. By comparison, the dorsal corticospinal axons were relatively richer in microtubules than the cuneate axons. Although the cuneate fiber tract contained many more large (greater than 2.0 microns 2 in cross section) axons than did the dorsal corticospinal tract, these differences in cytoskeletal organization were apparent even when myelinated axons of similar sizes (0.4 micron 2 to 2.0 microns 2) were compared. In addition, the number of neurofilaments in cuneate axons in the 0.4 to 2.0 microns 2 size range was significantly better correlated with axon size than was the case for this size range of dorsal corticospinal axons. Thus, the differences seen in the organization of the neurofilament latticework and the phosphorylation of NF-H between axons found in these two tracts both appeared to be correlated with cell type, and were independent of length or caliber of the axons.     

Specificity of human anti-neurofilament autoantibodies

The specificities and isotypes of human antibodies that react with neurofilament (NF) proteins were examined by Western blot analysis. Two-thirds of the subjects tested had antibodies to the 200 kDa high molecular weight neurofilament protein (NF-H), and fewer had antibodies to the low and middle molecular weight neurofilament proteins (NF-L and NF-M respectively). Human autoantibodies bound to both native and dephosphorylated NF-H, but some antibodies bound to dephosphorylated NF-H only, indicating the presence of at least two target epitopes. They also recognized a fusion protein containing a segment of the NF-H protein produced by a cDNA clone in Escherichia coli, indicating that they bind to unmodified peptide epitopes. The anti-NF-H antibodies were mostly IgG, but were frequently complexed to IgA or IgM antibodies, possibly with rheumatoid factor or anti-idiotypic activity. These characteristics of anti-NF-H antibodies are most consistent with a secondary immune response that is antigen driven and T-cell dependent.     

Developmental patterns of intermediate filament gene expression in the normal hamster brain

We have examined the patterns of expression of the major intermediate filament (IF) protein mRNAs during development of the hamster brain. Quantitative northern blotting was used to examine changes in the levels of mRNAs for the low, middle and high molecular weight neurofilament proteins (NF-L, NF-M, NF-H) as well as peripherin, vimentin and glial fibrillary acidic protein (GFAP). Total RNA was isolated from hamster brains at embryonic (E) days 12 and 14 and postnatal (P) days 1, 3, 5, 7, 9, 11, 13, 15, 20, 28 and 60-90 (adult), and probed with specific IF cDNAs. Northern blotting revealed that NF-L and NF-M mRNAs were present at very low levels in embryonic brain and that significant expression of these genes only occurred postnatally when the levels increased dramatically until P28 and then declined again in the adult. Increases in NF-H mRNA levels were somewhat delayed relative to those of NF-L and NF-M. NF-H mRNA was not seen at embryonic stages and was expressed at very low levels prior to P9; after that time the levels increased rapidly until P28 and then declined in the adult. Two of the type III IF genes, peripherin and vimentin, followed a pattern of expression opposite that of the NF genes. Both peripherin and vimentin mRNAs were present in embryonic brain and were expressed at higher levels during early postnatal stages than at later times. The magnitude and rate of reduction in vimentin gene expression in the postnatal interval was much greater than that of peripherin. GFAP mRNA levels were extremely low prior to P9 after which a robust increase occurred, followed by a decline in the adult. We discuss the implication of the dramatic changes in IF isotype expression in brain to the pathways of both neuronal and glial development in vivo.     

Transfected rat high-molecular-weight neurofilament (NF-H) coassembles with vimentin in a predominantly nonphosphorylated form

A fully encoding cDNA for the high-molecular-weight rat neurofilament protein (NF-H) has been isolated from a lambda gt11 library, sequenced and subcloned into eukaryotic expression vectors. Sequence analysis shows that rat NF-H has an overall homology of 72 and 88% with human and mouse NF-H, respectively. The head and rod domains are almost entirely identical, and the divergences are due to differences in the long C-terminal extensions of the molecule. The consensus phosphorylation sequence for neurofilaments Lys-Ser-Pro (KSP) is present 52 times. The predicted molecular mass of the protein is 115 kDa, 42% lower than that observed by SDS-PAGE. Upon transfection into vimentin-containing fibroblasts, such as L tk-, L929, and 3T6 cells, NF-H is seen distributed with vimentin by light and electron microscopic examinations indicating that copolymers of NF-H and vimentin are formed in these cells. Only a negligible proportion of the cells is positive when stained with a number of antibodies directed against phosphorylated NF-H epitopes. This is in contrast with the middle molecular weight NF protein (NF-M) transfected into L tk- and L929 cells, which can readily be detected by antibodies against phosphorylated neurofilament epitopes. The mobilities of the transfected protein on 1- and 2-dimensional gels confirm that NF-H is predominantly in a nonphosphorylated form. These results indicate that phosphorylation of NF-H, but not NF-M, on the KSP sequence is due to protein kinases, which are not present in fibroblasts and are presumably NF-H specific. The stable NF-H-expressing cell lines can therefore be used to study these putative neurofilament kinases in vitro and in vivo.     

Early in vitro genesis and differentiation of axons and dendrites by hippocampal neurons analyzed quantitatively with neurofilament-H and microtubule-associated protein 2 antibodies

Differentiating neurons initially extend neurites that are the precursors of axons and dendrites. The temporal pattern of neurite outgrowth has been studied extensively, but mostly qualitative analyses have been used to study this phenomenon. We have examined neurite outgrowth of hippocampal neurons in primary cultures using a polyclonal antibody against microtubule-associated protein 2 (MAP2) and a novel monoclonal antibody against the phosphorylated form of high neurofilament subunit (NF-H). These antibodies serve as markers for dendrites and axons, respectively. The neurite staining patterns were quantified during the first 10 days in culture and the analysis revealed that primary processes undergo three phases of differentiation: (i) in the first 24 h, the majority of primary neurites express MAP2 only and a small percentage express both MAP2 and NF-H; (ii) between 24 and 48 h, NF-H expression increases and it is coexpressed with MAP2 in many neurites as they begin to lengthen; and (iii) between 48 h and 4 days, MAP2 and NF-H protein expression occurs in separate populations of neurites. While most of the earliest forming primary neurites appear to be dendritic (MAP2 only), the coexpression of dendritic and axonal protein markers in a group of early forming processes suggests that these neurites may not be predetermined to become a dendrite or an axon. Our data also indicate that NF-H is detectable early in primary neurite development and that, based on in vivo localization and morphology of cultured neurites, the phosphorylated form of NF-H is concentrated in axons.     

Constitutive expression of the mature array of neurofilament proteins by a CNS neuronal cell line.

Neurofilament protein expression was examined immunochemically in a neuronal cell line derived from postnatal day 21 septal tissue. The SN48.1p cell line was found to constitutively synthesize an array of neurofilament proteins typical of a mature neuron. All three neurofilament subunits (NF-L, NF-M, and NF-H) as well as differentially phosphorylated isoforms (P-, P+, P++, and P ) of NF-M and NF-H were identified by immunoblot analysis. Immunofluorescence studies revealed that the neurofilament proteins were components of discrete, filamentous structures. Abnormal intracellular aggregations of neurofilament proteins were never observed. Some SN48.1p cells apportioned specific isoforms into selected intracellular regions based on the molecular weight and phosphorylation level of the protein. NF-L was preferentially localized to perikarya and proximal neurites; NF-M[P++] and NF-H[P ] were distributed to distal aspects of neurites. The expression of these differentiated features of neurofilament proteins and, presumably, the synthesis of the kinases and phosphatases required for normal neurofilament metabolism occurred in the absence of growth factors, differentiating agents, and specialized culture substrates. In addition, the non-neuronal intermediate filaments glial fibrillary acidic protein and epithelial cytokeratin proteins were absent. These data demonstrate that SN48.1p cells exhibit a neurofilament phenotype characteristic of mature neurons and provide a unique model to examine the expression and function of neurofilaments in differentiated neuronal cells.     

Development of the pyramidal tract in the hamster. II. An electron microscopic study

We undertook a qualitative and quantitative electron microscopic study of the growth and development of the pyramidal tract in the hamster to investigate the mode of growth of the axons, the possibility of fiber degeneration during development, and the process of myelination. By calculating the total fiber number as the product of axon density and tract area for several postnatal ages, we found that the pyramidal tract grows through the medulla as a compact bundle containing nearly twice the number of fibers as the mature tract. During the second postnatal week there is a substantial loss of axons followed in the third and fourth weeks by a more gradual loss such that by 34 days after birth the total number of axons reaches the adult value. Myelination in the hamster pyramidal tract begins at 7 days and continues at a very slow rate until the third postnatal week, when a dramatic increase in myelin formation occurs. By 34 days after birth the number of myelinated axons is approximately 80% that of the adult. As has been reported for other CNS tracts, there does not seem to be a “critical diameter” of an axon that absolutely determines the presence or absence of myelin on a fiber. However, all axons above 0.5 μm in diameter are myelinated at approximately the same rate, while those under this diameter are myelinated much more slowly and even in the adult make up only a small percentage of the total myelinated fibers.     

Biochemical and immunocytochemical characterization and distribution of phosphorylated and nonphosphorylated subunits of neurofilaments in squid giant axon and stellate ganglion

Monoclonal antibodies to squid neurofilament (aNFP) and intermediate filament (aIFA) proteins were used as probes for the biochemical and immunocytochemical analyses of neurofilament structure and distribution in the squid giant axon and stellate ganglion. On Western blots the aNFP antibody stained exclusively the 220 kDa and high-molecular-weight (HMW) components of neurofilaments in the giant axon, whereas the aIFA antibody primarily labeled the 60 kDa protein in the giant axon and the 60 and 65 kDa proteins in the stellate ganglion. Dephosphorylation of axoplasmic proteins by alkaline phosphatase resulted in a decrease in the molecular weights of both the 220 kDa and HMW neurofilament proteins and a concomitant loss of reactivity with the aNFP antibody on Western blots. This indicated that the aNFP antibody is specific for a phosphorylated epitope in the neurofilament. Increased dephosphorylation of the 220 kDa protein led to an enhanced immunostaining of the resultant 190 kDa polypeptide by the aIFA antibody, suggesting that the phosphorylated epitope may mask the conserved epitope recognized by aIFA. Light and electron microscopic immunocytochemical studies show intense labeling by the aNFP antibody in the giant axon. In contrast, the aIFA antibody labeled the glial cells around the giant axon intensely, while labeling of the giant axon itself was considerably less than that with the aNFP antibody. Since the 60 kDa protein in axoplasm is intensely stained by the aIFA antibody on Western blots, the relatively low amounts of labeling seen on semithin and thin sections of the giant axon by this antibody may be due to the masking of the 60 kDa protein by in situ fixed axoplasmic proteins. However, the aIFA antibody intensely labeled glial cells within the stellate ganglion and "islands" of filaments and nuclear membranes within ganglion cells. No reactivity for either antibody was seen in synapses. The aNFP antibody specifically labeled "beadlike" portions and cross-bridges on the axonal neurofilaments, suggesting that these components consist of the 220 kDa and HMW proteins. In contrast, the aIFA antibody labeled relatively smooth filaments in ganglion and glial cells. These data suggest that the 65 kDa protein represents the squid glial filament protein and that the 60 kDa protein found in axoplasm represents the low-molecular-weight subunit in the axonal neurofilament. The latter appears to be formed and/or organized in "islands" of filaments within ganglion cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential expression and modification of neurofilament triplet proteins during cat cerebellar development

Neurofilament (NF) proteins consist of three subunits of different molecular weights defined as NF-H, NF-M, and NF-L. They are typical structures of the neuronal cytoskeleton. Their immunocytochemical distribution during postnatal development of cat cerebellum was studied with several monoclonal and polyclonal antibodies against phosphorylated or unmodified sites. Expression and distribution of the triplet neurofilament proteins changed with maturation. Afferent mossy and climbing fibers in the medullary layer contained NF-M and NF-L already at birth, whereas NF-H appeared later. Within the first three postnatal weeks, all three subunits appeared in mossy and climbing fibers in the internal granular and molecular layers and in the axons of Purkinje cells. Axons of local circuit neurons such as basket cells expressed these proteins at the end of the first month, whereas parallel fibers expressed them last, at the beginning of the third postnatal month. Differential localization was especially observed for NF-H. Depending on phosphorylation, NF-H proteins were found in different axon types in climbing, mossy, and basket fibers or additionally in parallel fibers. A nonphosphorylated NF-H subunit was exclusively located in some Purkinje cells at early developmental stages and in some smaller interneurons later. A novel finding is the presence of a phosphorylation site in the NF-H subunit that is localized in dendrites of Purkinje cells but not in axons. Expression and phosphorylation of the NF-H subunit, especially, is cell-type specific and possibly involved in the adult-type stabilization of the axonal and dendritic cytoskeleton. © 1996 Wiley-Liss, Inc.     

Early posttranslational modifications of the three neurofilament subunits in mouse retinal ganglion cells: neuronal sites and time course in relation to subunit polymerization and axonal transport

We have characterized stages in the posttranslational processing of the three neurofilament subunits, High (NF-H), Middle (NF-M), and Low (NF-L), in retinal ganglion cells in vivo during the interval between synthesis in cell bodies within the retina and appearance of these polypeptides in axons at the level of the optic nerve (optic axons). Neurofilament proteins pulse-labeled by injecting mice intravitreally with [35S]methionine or [32P]orthophosphate, were isolated from Triton-soluble and Triton-insoluble fractions of the retina or optic axons by immunoprecipitation or immunoaffinity chromatography. Within 2 h after [35S]methionine injection, the retina contained neurofilament-immunoreactive radiolabeled proteins with apparent molecular weights of 160, 139, and 70 kDa, which co-migrated with subunits of axonal neurofilaments that were dephosphorylated in vitro with alkaline phosphatase. The two larger polypeptides were not labeled with [32P]orthophosphate, indicating that they were relatively unmodified forms of NF-H and NF-M. About 75% of the subunits were Triton-insoluble by 2 h after isotope injection, and this percentage increased to 98% by 6 h. Labeled neurofilament polypeptides appeared in optic axons as early as 2 h after injection. These subunits exhibited apparent molecular weights of 160, 139, and 70 kDa and were Triton-insoluble. The time of appearance of fully modified polypeptide forms differed for each subunit (2 h for NF-L, 6-18 h for NF-M, 18-24 h for NF-H) and was preceded by the transient appearance of intermediate forms. The modified radiolabeled subunits in optic axons 3 days after synthesis were heavily labeled with [32P]orthophosphate and exhibited the same apparent molecular weights as subunits of axonal neurofilaments (70 kDa, 145 and 140 kDa, and 195-210 kDa, respectively). Whole mounts of retina immunostained with monoclonal antibodies against NF-H in different states of phosphorylation demonstrated a transition from non-phosphorylated neurofilaments to predominantly phosphorylated ones within a region of the axon between 200 and 1000 microns downstream from the cell body. These experiments demonstrate that the addition of most phosphate groups to NF-M and NF-H takes place within a proximal region of the axon. The rapid appearance of modified forms of NF-L after synthesis may imply that processing of this subunit occurs at least partly in the cell body. The presence of a substantial pool of Triton-insoluble, unmodified subunits early after synthesis indicates that the heaviest incorporation of phosphate occurs after neurofilament proteins are polymerized.(ABSTRACT TRUNCATED AT 400 WORDS)     

Alzheimer's disease antibodies bind specifically to a neurofilament protein in Torpedo cholinergic neurons

Alzheimer's disease (AD) is characterized by neurofibrillary tangles and neuritic plaques and by the degeneration of central cholinergic neurons. Recent studies indicated the presence of antibodies in the sera and cerebrospinal fluid of AD patients which react with neuronal tissue and which recognize cholinergic neurons. In order to identify the cholinergic antigens against which the AD antibodies are directed, we have recently used the purely cholinergic electromotor neurons of the electric fish Torpedo which are chemically homogenous and cross-react antigenically with mammalian cholinergic neurons. This study revealed that immunoglobulins (IgG) from sera of AD patients bind specifically to an antigen in Torpedo electromotor neurons with an apparent molecular weight of 200 kDa. In the present report we attempt to characterize this antigen. The similarity in size of this protein to that of the heavy neurofilament subunit (NF-H) and the association of neurofilaments with plaques and tangles prompted us to examine the possibility that it is a neurofilament protein. Our findings show that IgG from sera of AD patients bind to the NF-H protein of Torpedo cholinergic neurons. Comparison of the binding of AD and control IgG to Torpedo cholinergic NF-H revealed that AD IgG bind to this neurofilament protein more readily than do control IgG. In contrast, AD and control IgG bind similarly to NF-H obtained from the chemically heterogenous Torpedo spinal cord and rat brain. These findings suggest that AD sera contain a repertoire of anti-NF-H IgG and that a subpopulation of these antibodies whose levels are significantly elevated in AD binds to epitopes highly enriched in Torpedo cholinergic NF-H.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of stage-specific neurite-associated proteins in the developing murine nervous system recognized by a monoclonal antibody

A monoclonal antibody, 4D7, obtained with embryonic rat brain as an immunogen, recognizes an epitope on 3 protein species of 150-160, 100-110, and 80 kDa, present in mouse and rat brain during the fetal period. Vital immunostaining of dissociated cultures of fetal forebrain indicates that the antigen is localized largely on the external plasma membrane of a subpopulation of neurites. Immunocytochemistry reveals that the distribution of the antigen in vivo is restricted to the nervous system. Immunoreactivity is concentrated primarily in the pathways of a limited set of CNS and PNS axon systems during early stages of their development, as delineated by staining with the neurofilament antibody, C2. Depending on the particular axon system, immunoreactivity with 4D7 persists only for one to several days of prenatal or perinatal development. In the spinal cord, stage-specific-neurite-associated proteins (SNAP) expression occurs first along motor axon pathways on embryonic day (E) 10 and then within the nerve trunks of dorsal root ganglia and the commissural fiber system on E11. Immunoreactivity is detectable among most cranial nerves starting in the interval from E11 to E13. Within the brain, the onset of SNAP expression within several discrete axon tracts occurs in the interval E14-16, including the lateral olfactory tract, anterior commissure, corpus callosum, fasciculus retroflexus, and fornix. Immunoreactivity within the embryonic intermediate zones of some structures matches the location of certain other axon systems. Sites of 4D7 staining which do not correspond to the location of axon populations include the internal portion of the external granular layer of the postnatal cerebellum and the cortex of the reeler mutant mouse. The predominant localization of the 4D7 antigen among axon systems and its precisely regulated spatio-temporal pattern of expression are consistent with the possibility that the SNAP antigens play a significant role in the early stages of growth of axonal tracts in vivo.     

Differential expression and localization of the phosphorylated and nonphosphorylated neurofilaments during the early postnatal development of rat hippocampus

Neurofilament (NF) proteins are expressed in most mature neurons in the central nervous system. Although they play a crucial role in neuronal growth, organization, shape, and plasticity, their expression pattern and cellular distribution in the developing hippocampus remain unknown. In the present study, we have used Western blotting and immunocytochemistry to study the low- (NF-L), medium- (NF-M), and high- (NF-H) molecular-weight NF proteins; phosphorylated epitopes of NF-M and NF-H; and a nonphosphorylated epitope of NF-H in the early postnatal (through P1-P21) development of the rat hippocampus. During the first postnatal week, NF-M was the most abundantly expressed NF, followed by NF-L, whereas the expression of NF-H was very low. Through P7-P14, the expression of NF-H increased dramatically and later began to plateau, as also occurred in the expression of NF-M and NF-L. At P1, no NF-M immunopositive cell bodies were detected, but cell processes in the CA1-CA3 fields were faintly immunopositive for NF-M and for the phosphorylated epitopes of NF-M and NF-H. At P7, CA3 pyramidal neurons were strongly immunopositive for NF-L and NF-H, but not for NF-M. The axons of granule cells, the mossy fibers (MFs), were NF-L and NF-M positive through P7-P21 but were NF-H immunonegative at all ages. Although they stained strongly for the phosphorylated NF-M and NF-H at P7, the staining intensity sharply decreased at P14 and remained so at P21. The cell bodies of CA1 pyramidal neurons and granule cells remained immunonegative against all five antibodies in all age groups. Our results show a different time course in the expression and differential cell type and cellular localization of the NF proteins in the developing hippocampus. These developmental changes could be of importance in determining the reactivity of hippocampal neurons in pathological conditions in the immature hippocampus.     

Inhibition of axonal development after injection of neurofilament antibodies into a Xenopus laevis embryo

The ability to target specific cytoskeletal components in axons for disruption within intact developing embryos would provide a valuable tool for studying neuronal development. Neurofilaments are an attractive target for such an approach, because they are neuron specific and are expressed late in embryogenesis principally beginning during axon outgrowth. No pharmacological agents are currently available that disrupt neurofilaments without also affecting general development. One approach that has been used successfully to affect proteins in vivo is to inject specific antibodies into living cells. We employed this approach in Xenopus laevis embryos by injecting two antibodies directed against the middle molecular weight neurofilament protein (NF-M) into a single blastomere of a two-cell stage embryo. Injected antibodies could be detected for as long as 3.5 days in cells descended from the injected blastomere. Only cell bodies of neurons descended from anti-NF-M-injected blastomeres contained abnormal accumulations of intermediate filament proteins, and peripheral nerve development was unilaterally retarded in these neurofilament antibody-injected tadpoles. Such accumulations and peripheral nerve defects were not seen in neurons derived from uninjected blastomeres or from blastomeres injected with control antibodies. These data demonstrate the usefulness of specific antibodies to perturb neuronal development in intact frog embryos and, in addition, suggest a role for neurofilaments in axon elongation.     

Axonal cytoskeletal pathology in aged and diabetic human sympathetic autonomic ganglia

Prevertebral sympathetic ganglia develop markedly enlarged argyrophilic neurites as a function of age, gender and diabetes. Immunolocalization studies demonstrate their preferential labeling with antisera to highly phosphorylated 200 kDa neurofilament (NF-H) epitopes, NPY, peripherin and synapsin I, but not to hypophosphorylated NF-M and NF-H or MAP-2. The immunophenotype of dystrophic neurites in conjunction with the results of histochemical and ultrastructural studies are consistent with the terminal axonal and/or synaptic origin of neuritic dystrophy in the sympathetic ganglia of aged and diabetic human subjects.     

Selective regulation of axonal growth from developing hippocampal neurons by tumor necrosis factor superfamily member APRIL

APRIL (A Proliferation-Inducing Ligand, TNFSF13) is a member of the tumor necrosis factor superfamily that regulates lymphocyte survival and activation and has been implicated in tumorigenesis and autoimmune diseases. Here we report the expression and first known activity of APRIL in the nervous system. APRIL and one of its receptors, BCMA (B-Cell Maturation Antigen, TNFRSF17), are expressed by hippocampal pyramidal cells of fetal and postnatal mice. In culture, these neurons secreted APRIL, and function-blocking antibodies to either APRIL or BCMA reduced axonal elongation. Recombinant APRIL enhanced axonal elongation, but did not influence dendrite elongation. The effect of APRIL on axon elongation was inhibited by anti-BCMA and the expression of a signaling-defective BCMA mutant in these neurons, suggesting that the axon growth-promoting effect of APRIL is mediated by BCMA. APRIL promoted phosphorylation and activation of ERK1, ERK2 and Akt and serine phosphorylation and inactivation of GSK-3β in cultured hippocampal pyramidal cells. Inhibition of MEK1/MEK2 (activators of ERK1/ERK2), PI3-kinase (activator of Akt) or Akt inhibited the axon growth-promoting action of APRIL, as did pharmacological activation of GSK-3β and the expression of a constitutively active form of GSK-3β. These findings suggest that APRIL promotes axon elongation by a mechanism that depends both on ERK signaling and PI3-kinase/Akt/GSK-3β signaling.     

Expression of neuronal and glial polypeptides during histogenesis of the human cerebellar cortex including observations on the dentate nucleus

In order to gain a more complete understanding of the sequential pattern of gene expression during neurogenesis and gliogenesis in humans, we followed the expression of well-characterized, developmentally regulated polypeptides in the cerebellar cortex and dentate nucleus by immunohistochemistry using monoclonal antibodies of highly defined specificity. At 8–10 weeks gestational age (GA), progenitor cells and their immediate progeny in the rhombencephalic ventricular zone expressed vimentin and nestin and, to a lesser extent, microtubule-associated protein 5 (MAP5) and glial fibrillary acidic protein (GFAP), but not the low affinity nerve growth factor receptor (NGFR). In contrast, postmitotic, migrating immature neurons in the intermediate zone gave strong reactions for MAP2, tau, and a nonphosphorylated form of middle molecular weight neurofilament (NF) protein (NF-M) and weak reactivity for NGFR. At 15 weeks GA, proliferating cells of the superficial part of the cerebellar external granular layer stained only for NGFR, while more deeply situated cells of the external granular layer stained positively for NGFR, MAP2, MAP5, tau, and chromogranin A, which correlates with the early outgrowth of parallel fibers. All phosphoisoforms of NF-M as well as the low (NF-L) and high (NF-H) molecular weight NF proteins and alpha-internexin were expressed in the somatodendritic domain of Purkinje cells and dentate nucleus neurons from about 20 weeks GA with a gradual compartmentalization of highly phosphorylated forms of NF-M and NF-H into axons by the end of gestation. Alpha-internexin was also expressed strongly in axons of the deep white matter from 20 weeks GA to adulthood. MAP2, synaptophysin, and NGFR showed early, transient expression in the somatodendritic domain of Purkinje cells followed by the appearance of a 220 kDa nestin-like peptide that continued to be expressed in adult Purkinje cells. Notably, developing dentate nucleus neurons expressed many of these proteins in a similar temporal sequence. Early in the developing cerebellar cortex, the expression of NF protein and synaptophysin occurred in discrete patches or columns similar to those described for other antigens (i.e., zebrins). Finally, radial glia were positive for vimentin, GFAP, and nestin from 8 weeks GA to 8 months postnatal. This study describes the distinct molecular programs of lineage commitment in cerebellar progenitor cells and in differentiating neurons and astrocytes of the human cerebellum. The acquisition of a mature molecular neuronal phenotype correlates with the establishment of structural polarity in cerebellar neurons. © 1993 Wiley-Liss, Inc.