Microtubule associated proteins (MAPs) interact with tubulin to modulate neurite stability and growth during development. The phosphorylated form of one of these MAPs, MAP1B (MAP1B-P) is hypothesized to be of particular importance for the regulation of neurite outgrowth. To investigate the mechanisms by which MAP1B and MAP1B-P contribute to this regulation, we used a new antibody against an isoform of MAP1B-P to determine its pattern of expression during neuronal development in vitro. We examined cultured hippocampal neurons because these provide a well-established system to evaluate the development of axons and dendrites. MAP1B, MAP1B-P and MAP2 colocalized to the cell bodies and minor processes during the first 24 hours of culture, but MAP1B-P also extended well into the growth cones. As neurite outgrowth and differentiation proceeded, MAP1B and MAP1B-P became localized to the cell bodies and axons, and MAP2 to the cell bodies and dendrites. After 3 days, MAP1B-P declined in the cell body and was segregated to the distal axon; MAP1B remained in the cell body, but was also concentrated in the distal axon. Over 5-9 days in culture, MAP1B-P levels decreased and became undetectable; MAP1B levels decreased later (19-23 days). MAP2 levels, however, remained high through the entire culture period in cell bodies and dendrites. These results are consistent with the hypothesis that MAP1B-P plays an important role in the initiation and elongation of axons by regulating the dynamics of microtubules near the growth cone: MAP1B-P expression is greatest during the period of active neurite extension, is particularly prominent in growth cones where axon outgrowth is most active, and decreases along with the decline in active axon extension.

The diversity of neuronal morphology and function is correlated with specific expression of various microtubule associated proteins (MAPs). One of the major neuronal MAPs, tau, has multiple isoforms formed as a result of alternative splicing and phosphorylation that are differentially expressed during development. Big tau is a high molecular weight isoform that contains an additional large exon (4a) and is expressed primarily by neurons in the peripheral nervous system (PNS). We cloned the complete 4a exon in an expression vector, isolated the recombinant protein and produced antibodies specific to Big tau that were used to localize Big tau in the developing spinal cord and in the adult central nervous system (CNS). In developing spinal cord, Big tau is first expressed in the central projections of the dorsal root ganglia neurons and in motor neurons at embryonic day 18 and postnatal day 2, respectively. In the adult rat CNS, almost all neurons that extend processes into the PNS express Big tau, including all cranial nerve motor nuclei and central processes of most sensory ganglia; of these ganglia, only the bipolar neurons of the olfactory, vestibular and spiral ganglia did not express Big tau. Retinal ganglion cells are the only CNS neurons, whose processes remain entirely within the CNS, that express high levels of Big tau. The limited and specific distribution of Big tau is consistent with a role in stabilizing microtubules in axons that are subjected to great shear forces.

Vimentin is expressed initially by nearly all neuronal precursors in vivo, and is replaced by neurofilaments shortly after the immature neurons become post-mitotic. Moreover, both vimentin and neurofilaments can be detected transiently within the same neurite, leaving open the possibility that vimentin may play a role in the early stages of neuritogenesis. In the present study, cultured hippocampal neurons, which transiently express vimentin in culture, were treated with sense- and antisense-oriented deoxyoligonucleotides encoding regions of the vimentin sequence that overlap the translation initiation codon. Antisense oligonucleotide treatment reduced vimentin-immunoreactivity to background levels. Moreover, while 90-100% of cultured hippocampal neurons elaborated neurites within the first 24 hr following plating, only 24-30% did so in the presence of vimentin antisense oligonucleotides. Inhibition of neurite outgrowth was reversible following removal of antisense oligonucleotide. These findings substantiate earlier studies in neuroblastoma cells, indicating a possible role for vimentin in the initiation of neurite outgrowth.

The purpose of this investigation was to identify and localize tissue transglutaminase (TGase) within neurons from the hippocampi of normal aged individuals and of those with confirmed Alzheimer's disease (AD). This enzyme may be a factor in the molecular mechanisms of neurodegeneration and formation of insoluble macromolecular complexes found in the neurons of normal aged and AD brain tissue. An antibody made to the extracellular TGase, coagulation factor XIIIa, was found to be specific for purified intracellular guinea pig liver tissue TGase. The specificity for liver tissue TGase has enabled us to identify tissue TGase(s) within rat hippocampal neurons and within neurons from normal aged and AD hippocampal tissues. Degenerating neurons from the AD hippocampus, compared to neurons from the normal aged hippocampus, exhibited increased immunoreactivity for TGase and demonstrated co-labeling for PHF1 and anti-TGase. Our results suggest that TGase may be associated with the neurofibrillary degeneration observed in AD, thereby implicating TGase as a potential factor in the pathogenesis of Alzheimer's disease.

MAP1B is a major cytoskeletal protein in growing axons and is strongly regulated during brain development. The present studies compare the expression of MAP1B mRNA, the protein, and its phosphorylated isoform in spinal cord and dorsal root ganglia (DRGs) with brain. In spinal cord and brain, MAP1B mRNA levels were highest in early stages of development, decreased several fold during postnatal development, and remained low in adults. In contrast, there were no significant changes of MAP1B mRNA levels during development of DRG and they remained high in adults. The levels of MAP1B protein decreased in brain and spinal cord in parallel with the changes of their mRNA. The protein levels in DRG remained relatively high but declined in the sciatic nerve. Phosphorylated MAP1B was expressed in high levels during the early stages of development in brain, spinal cord, and sciatic nerve and decreased rapidly to undetectable levels postnatally except for sciatic nerve where it remained detectable. Immunohistochemical analysis showed that phosphorylated MAP1B was absent from DRG cell bodies at all stages but was present in axons of DRG and motor neurons in both spinal cord and sciatic nerve. Immunostaining also confirmed Western blot analysis indicating that MAP1B was initially abundant within the spinal cord but was at later stages present only in motor neurons and the central processes of DRG neurons. These results reflect differential distribution of MAP1B isoforms at different stages of development and in different regions of the nervous system.