The leukocyte common antigen-related protein tyrosine phosphatase receptor regulates regenerative neurite outgrowth in vivo.

Drosophila and leech models of nervous system development demonstrate that protein tyrosine phosphatase (PTP) receptors regulate developmental neurite outgrowth. Whether PTP receptors regulate neurite outgrowth in adult systems or in regenerative states remains unknown. The leukocyte common antigen-related (LAR) receptor is known to be present in rodent dorsal root ganglion (DRG) neurons; therefore, the well established model of postcrush sciatic nerve regeneration was used to test the hypothesis that LAR is required for neurite outgrowth in the adult mammalian nervous system. In uninjured sciatic nerves, no differences in nerve morphology and sensory function were detected between wild-type and LAR-deficient littermate transgenic mice. Sciatic nerve crush resulted in increased LAR protein expression in DRG neurons. In addition, nerve injury led to an increase in the proportion of LAR protein isoforms known to have increased binding affinity to neurite-promoting laminin-nidogen complexes. Two weeks after nerve crush, morphological analysis of distal nerve segments in LAR-deficient transgenic mice demonstrated significantly decreased densities of myelinated fibers, decreased axonal areas, and increased myelin/axon area ratios compared with littermate controls. Electron microscopy analysis revealed a significant twofold reduction in the density of regenerating unmyelinated fibers in LAR-/- nerves distal to the crush site. Sensory testing at the 2 week time point revealed a corresponding 3 mm lag in the proximal-to-distal progression of functioning sensory fibers along the distal nerve segment. These studies introduce PTP receptors as a major new gene family regulating regenerative neurite outgrowth in vivo in the adult mammalian system.

Nerve growth factor (NGF) loop 4 dimeric mimetics activate ERK and AKT and promote NGF-like neurotrophic effects.

Previous work indicating that nerve growth factor (NGF) protein loops 2 and 4 interact with TrkA receptors raise the possibility that small molecule mimetics corresponding to TrkA-interacting domains that have NGF agonist activity can be developed. We applied our previously developed strategy of dimeric peptidomimetics to address the hypothesis that loop 4 small molecule dimeric mimetics would activate TrkA-related signal transduction and mimic NGF neurotrophic effects in a structure-specific manner. A loop 4 cyclized peptide dimer demonstrated NGF-like neurotrophic activity, whereas peptides with scrambled sequence, added or substituted residues, or cyclized in monomeric form were inactive. Activity was blocked by the TrkA inhibitors K252a and AG879 but not by NGF p75 receptor blocking antibody. Dimeric, but not monomeric, peptides partially blocked NGF activity. This profile was consistent with that of a NGF partial agonist. ERK and AKT phosphorylation was stimulated only by biologically active peptides and was blocked by K252a. The ERK inhibitor U0126 blocked the neurite- but not the survival-promoting activity of both NGF and active peptide. These studies support the proof of concept that small molecule NGF loop 4 mimetics can activate NGF signaling pathways and can mimic death-preventing and neurite-promoting effects of NGF. This finding will guide the rational design of NGF single-domain mimetics and contribute to elucidating NGF signal transduction mechanisms.

Downregulation of LAR tyrosine phosphatase prevents apoptosis and augments NGF-induced neurite outgrowth.

The identity of the protein tyrosine phosphatases (PTPs) regulating cell death and responses to neurotrophins during neural development remain unknown. To determine if the leukocyte common antigen-related (LAR) PTP regulates these processes, PC12 cells were made LAR-deficient via stable transfection with an LAR antisense transgene. LAR-deficient cells demonstrated a stable novel phenotype, including a two-fold increase in nerve growth factor- but not fibroblast growth factor-induced neurite outgrowth. Upon serum-deprivation, LAR-deficient cells exhibited a two- to three-fold decrease in cell death. The findings that an endogenous PTP promotes cell death and counter-regulates neurotrophin actions introduce a major new receptor gene family to neurotrophic processes and suggest novel strategies for preventing cell death and augmenting neurotrophin function.

LAR tyrosine phosphatase receptor: proximal membrane alternative splicing is coordinated with regional expression and intraneuronal localization.

Examination of null-mutant Drosophila and Leukocyte Common Antigen-Related (LAR)-deficient transgenic mice has demonstrated that the LAR protein tyrosine phosphatase (PTP) receptor promotes neurite outgrowth. In the absence of known ligands, the mechanisms by which LAR-type PTP receptors are regulated are unknown. We hypothesized that an alternatively spliced eleven amino acid proximal membrane segment of LAR (LAR alternatively spliced element-a; LASE-a) contributes to regulation of LAR function. Human, rat and mouse LAR cDNA sequences demonstrated that the predicted eleven amino acid inserts in rat and mouse are identical and share nine of eleven residues with the human insert. LASE-a splicing led to the introduction of a Ser residue into LAR at a position analogous to Ser residues undergoing regulated phosphorylation in other PTPs. In-situ studies revealed increasingly region-specific expression of LASE-a containing LAR transcripts during postnatal development. RT-PCR analysis of cortical and hippocampal tissue confirmed that the proportion of LAR transcripts containing LASE-a decreases during development. Immunostaining of cultured PC12 cells, cerebellar granule neurons, dorsal root ganglia and sciatic nerve sections with antibody directed against the LASE-a insert demonstrated signal in cell bodies but little if any along neurites. In contrast, staining with antibody directed to a separate domain of LAR showed accumulation of LAR along neurites. The findings that LASE-a splicing is conserved across human, rat and mouse, that the LASE-a insert introduces a Ser at a site likely to be targeted for regulated phosphorylation and that developmentally regulated splicing is coordinated with specific regional and intraneuronal localization point to important novel potential mechanisms regulating LAR-type tyrosine phosphatase receptor function in the nervous system.