Tau proteins with frontotemporal dementia-17 mutations have both altered expression levels and phosphorylation profiles in differentiated neuroblastoma cells.

The inherited form of frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) has been attributed to mutations in the tau gene. Pathologically, affected FTDP-17 brains share tau aggregates with other tauopathies, the most common being Alzheimer's disease. FTDP-17 mutations may therefore affect tau function leading to tau aggregation and cell loss. Interaction of tau with microtubules is thought to be regulated by phosphorylation. Investigating FTDP-17 mutations transiently expressed as enhanced green fluorescent protein (EGFP)-tagged proteins for the first time in differentiated neuronal cells, we found that two out of three missense mutations showed surprisingly decreased phosphorylation at the pathologically relevant S202/T205 site, mutant EGFP-tau being completely dephosphorylated in most cells. Moreover, phosphorylation at the S396/S404 site was moderately decreased for all mutant isoforms. Although microtubule integrity was not affected, with all mutants tested we demonstrated an increase in cellular tau protein level, some of which is microtubule-bound. Further enhancing this EGFP-tau accumulation by inhibition of tau degradation resulted in the previously less phosphorylated mutant EGFP-tau becoming highly phosphorylated. We conclude that the missense tau mutations primarily result in an excess of neuronal tau, which may interfere with important cellular functions such as axonal transport.

Wallerian degeneration of injured axons and synapses is delayed by a Ube4b/Nmnat chimeric gene.

Axons and their synapses distal to an injury undergo rapid Wallerian degeneration, but axons in the C57BL/WldS mouse are protected. The degenerative and protective mechanisms are unknown. We identified the protective gene, which encodes an N-terminal fragment of ubiquitination factor E4B (Ube4b) fused to nicotinamide mononucleotide adenylyltransferase (Nmnat), and showed that it confers a dose-dependent block of Wallerian degeneration. Transected distal axons survived for two weeks, and neuromuscular junctions were also protected. Surprisingly, the Wld protein was located predominantly in the nucleus, indicating an indirect protective mechanism. Nmnat enzyme activity, but not NAD+ content, was increased fourfold in WldS tissues. Thus, axon protection is likely to be mediated by altered ubiquitination or pyridine nucleotide metabolism.

A Ufd2/D4Cole1e chimeric protein and overexpression of Rbp7 in the slow Wallerian degeneration (WldS) mouse.

Exons of three genes were identified within the 85-kilobase tandem triplication unit of the slow Wallerian degeneration mutant mouse, C57BL/Wld(S). Ubiquitin fusion degradation protein 2 (Ufd2) and a previously undescribed gene, D4Cole1e, span the proximal and distal boundaries of the repeat unit, respectively. They have the same chromosomal orientation and form a chimeric gene when brought together at the boundaries between adjacent repeat units in Wld(S). The chimeric mRNA is abundantly expressed in the nervous system and encodes an in-frame fusion protein consisting of the N-terminal 70 amino acids of Ufd2, the C-terminal 302 amino acids of D4Cole1e, and an aspartic acid formed at the junction. Antisera raised against synthetic peptides detect the expected 43-kDa protein specifically in Wld(S) brain. This expression pattern, together with the previously established role of ubiquitination in axon degeneration, makes the chimeric gene a promising candidate for Wld. The third gene altered by the triplication, Rbp7, is a novel member of the cellular retinoid-binding protein family and is highly expressed in white adipose tissue and mammary gland. The whole gene lies within the repeat unit leading to overexpression of the normal transcript in Wld(S) mice. However, it is undetectable on Northern blots of Wld(S) brain and seems unlikely to be the Wld gene. These data reveal both a candidate gene for Wld and the potential of the Wld(S) mutant for studies of ubiquitin and retinoid metabolism.

The microtubule-associated protein MAP1B is involved in local stabilization of turning growth cones.

For the development of the nervous system it is crucial that growth cones detect environmental information and react by altering their growth direction. The latter process is thought to depend on local stabilization of growth cone microtubules. We have obtained evidence of a role for the microtubule-associated protein MAP1B, in particular a mode 1 phosphoisoform of the molecule, P1-MAP1B, in this process. P1-MAP1B is tightly associated with the cytoskeleton and is present at highest concentrations in the distal axon and the growth cone of chick retinal ganglion cells. In growth cones turning at nonpermissive substrate borders, P1-MAP1B is restricted to regions which are stabilized. Unilateral neutralization of P1-MAP1B in one-half the growth cone by microscale chromophore-assisted laser inactivation changes growth cone motility, morphology, and growth direction. The results indicate a functional role for P1-MAP1B in local growth cone stabilization and thus growth cone steering.

Mutations in tau reduce its microtubule binding properties in intact cells and affect its phosphorylation.

In vitro evidence has suggested a change in the ability of tau bearing mutations associated with fronto-temporal dementia to promote microtubule assembly. We have used a cellular assay to quantitate the effect of both isoform differences and mutations on the physiological function of tau. Whilst all variants of tau bind to microtubules, microtubule extension is reduced in cells transfected with 3-relative to 4-repeat tau. Mutations reduce microtubule extension with the P301L mutation having a greater effect than the V337M mutation. The R406W mutation had a small effect on microtubule extension but, surprisingly, tau with this mutation was less phosphorylated in intact cells than the other variants.

Two strategies to prepare neural cortical cytoskeleton components for the generation of monoclonal antibodies.

Like most other cells, neurons possess a spectrin/actin based network closely associated with the inner side of the cell membrane, the cortical cytoskeleton. This structure serves many diverse functions during axonal outgrowth. In the growth cone, the cortical cytoskeleton is involved in surface shaping, modulation of integral membrane proteins, and signal transduction. We developed two strategies to prepare material enriched for neural cortical cytoskeleton. The first strategy combined the isolation of a membrane/cortical cytoskeleton fraction by density gradient centrifugation with an enzymatic degradation of cell surface proteins. The second strategy is based on the attachment and crosslinking of single cells to beads, allowing for the removal of the cell contents by cell disruption; only membrane/cortical cytoskeleton patches are retained on the beads. Both strategies made use of the intimate association of the cortical cytoskeleton with the cell membrane, permitting the removal of cytoplasm, organelles and cytoplasmic cytoskeleton while retaining the cortical cytoskeleton. Monoclonal antibodies generated using both preparations as immunization material were screened for recognition of intracellular structures in axons and growth cones of retinal ganglion cells in culture. A quantitative specification of the antibodies is presented and six antibodies are characterized in immunolabelings and Western blot analysis.

Cell adhesion molecule SC1/DMGRASP is expressed on growing axons of retina ganglion cells and is involved in mediating their extension on axons.

We determined expression and function of a cell membrane protein in the developing chick retinotectal system identified by a monoclonal antibody (mAb 4H5) and the corresponding antiserum. Our data revealed that the protein shares a series of properties, including the N-terminal amino acid sequence, with a cell adhesion molecule termed DM-GRASP, SC1, BEN, and JC7. It can therefore be considered identical with this molecule and is referred to as SC1/DMGRASP. In early development of the retinotectal system, SC1/DMGRASP is exclusively expressed on growing, far-projecting, tract-forming axons. Expression begins at the onset of retina ganglion cell axogenesis and its maximum overlaps with the phase of maximal axon extension. Later in development, SC1/DMGRASP appears on distinct laminae within plexiform layers in spatiotemporal correlation with synaptogenesis. In an in vitro assay system designed to study the elongation of RGC axonal processes on preexisting RGC axons, addition of SC1/DMGRASP antiserum specifically reduces lengths of axonal processes. In contrast, axonal growth on laminin or basal lamina preparations is not SC1/DMGRASP-dependent. Taken together, the data provide evidence for a role of SC1/DMGRASP in axonal elongation of SC1/DMGRASP-positive axons on such axons, thereby possibly contributing to the pathway and target finding mechanisms of far-projecting, tract-forming central nervous system neurons.