Dusan Matusica

Proteolytic processing of the p75 neurotrophin receptor: A prerequisite for signalling?: Neuronal life, growth and death signalling are crucially regulated by intra-membrane proteolysis and trafficking of p75(NTR)

The common neurotrophin receptor (p75NTR) regulates various functions in the developing and adult nervous system. Cell survival, cell death, axonal and growth cone retraction, and regulation of the cell cycle can be regulated by p75NTR‐mediated signals following activation by either mature or pro‐neurotrophins and in combination with various co‐receptors, including Trk receptors and sortilin. Here, we review the known functions of p75NTR by cell type, receptor‐ligand combination, and whether regulated intra‐membrane proteolysis of p75NTR is required for signalling. We highlight that the generation of the intracellular domain fragment of p75NTR is associated with many of the receptor functions, regardless of its ligand and co‐receptor interactions.

Characterization and use of the NSC‐34 cell line for study of neurotrophin receptor trafficking

This study addressed the suitability of the NSC‐34 cell line as a motor neuron‐like model for investigating neurotrophin receptor trafficking and associated subcellular processes. Initially, culture conditions were optimized for the use of NSC‐34 cells in confocal microscopy. Cell surface markers, as well as markers associated with the regulated endosomal pathway thought to be associated with neurotrophin receptor transport, were identified. The study revealed the presence of a number of molecules previously not described in the literature, including the tropomyosin‐like receptor kinase C (TrkC), sortilin, the vesicular acetylcholine transporter (VAChT), and the lipid raft‐associated ganglioside GT1b. The presence of both sortilin and Gt1b was of special interest, insofar as these markers have been implicated in direct relationships with the p75NTR receptor. Evidence is provided for neurotrophin‐dependent internalization of p75NTR and TrkB. Both nerve growth factor (NGF) and brain‐derived neurotrophic factor (BDNF) increased the rate of internalization of p75NTR, with internalization dynamics comparable to those described for other cell lines. Thus, these studies not only describe components of the regulatory process governing the trafficking of this important receptor but also clearly demonstrate the value of NSC‐34 cells as a suitable motor neuron model for the study of internalization and trafficking of cell surface molecules.

ProNGF mediates death of Natural Killer cells through activation of the p75NTR–sortilin complex

The common neurotrophin receptor P75NTR, its co-receptor sortilin and ligand proNGF, have not previously been investigated in Natural Killer (NK) cell function. We found freshly isolated NK cells express sortilin but not significant amounts of P75NTR unless exposed to interleukin-12 (IL-12), or cultured in serum free conditions, suggesting this receptor is sequestered. A second messenger associated with p75NTR, neurotrophin-receptor-interacting-MAGE-homologue (NRAGE) was identified in NK cells. Cleavage resistant proNGF123 killed NK cells in the presence of IL-12 after 20h and without IL-12 in serum free conditions at 48h. This was reduced by blocking sortilin with neurotensin. We conclude that proNGF induced apoptosis of NK cells may have important implications for limiting the innate immune response.

Functional monoclonal antibodies to p75 neurotrophin receptor raised in knockout mice

In this study, p75NTREXONIII knockout mice were used as immune-naive hosts to produce functional antibodies to human p75NTR. Three monoclonal antibodies were produced and named MLR1, MLR2 and MLR3, and isotyped as IgG1, IgG2a and IgG2a, respectively. MLR1 and MLR2 bound to human p75NTR with higher affinity than the well-characterized ME20.4 in ELISA and also recognized p75NTR present on neurons in both rat and mouse. MLR1 and MLR2 bound to nerves known to express p75NTR following injection into Balb/C mice but not p75NTREXONIII knockout mice, indicating the antibodies are directed against the ligand binding extracellular region absent in knockout mice. Both MLR1 and MLR2 partially blocked NGF induced cell death in a mouse cell-line that expresses p75NTR but not TrKA. Importantly, intracerebroventricular injections indicated MLR2 was internalized within the cell bodies of mouse basal forebrain neurons, further demonstrating that this antibody is biologically active.

Mapping of the interaction site between sortilin and the p75 neurotrophin receptor reveals a regulatory role for the sortilin intracellular domain in p75 neurotrophin receptor shedding and apoptosis.

Background: Sortilin and p75NTR induce neuronal apoptosis by binding pro-neurotrophins during development and following neuronal injury. Results: Sortilin interacts with an extracellular juxtamembrane 23-amino acid sequence of p75NTR. Conclusion: Despite binding being mediated through extracellular interactions, the intracellular domain of sortilin regulates p75NTR shedding and apoptosis. Significance: Mapping may allow design of compounds inhibiting neuronal cell death by blocking the interaction between sortilin and p75NTR. Neurotrophins comprise a group of neuronal growth factors that are essential for the development and maintenance of the nervous system. However, the immature pro-neurotrophins promote apoptosis by engaging in a complex with sortilin and the p75 neurotrophin receptor (p75NTR). To identify the interaction site between sortilin and p75NTR, we analyzed binding between chimeric receptor constructs and truncated p75NTR variants by co-immunoprecipitation experiments, surface plasmon resonance analysis, and FRET. We found that complex formation between sortilin and p75NTR relies on contact points in the extracellular domains of the receptors. We also determined that the interaction critically depends on an extracellular juxtamembrane 23-amino acid sequence of p75NTR. Functional studies further revealed an important regulatory function of the sortilin intracellular domain in p75NTR-regulated intramembrane proteolysis and apoptosis. Thus, although the intracellular domain of sortilin does not contribute to p75NTR binding, it does regulate the rates of p75NTR cleavage, which is required to mediate pro-neurotrophin-stimulated cell death.

An intracellular domain fragment of the p75 neurotrophin receptor (p75(NTR)) enhances tropomyosin receptor kinase A (TrkA) receptor function.

Background: The mechanism by which the p75 neurotrophin receptor (p75NTR) and TrkA interact to enhance neurotrophin signaling is unknown. Results: The p75NTR intracellular domain fragment, p75ICD, but not full-length p75NTR enhanced NGF binding to TrkA and neurite outgrowth. Conclusion: The results suggest that p75ICD causes a conformational change within the extracellular domain of TrkA. Significance: The findings challenge our current understanding of how p75NTR enhances neurotrophic activity. Facilitation of nerve growth factor (NGF) signaling by the p75 neurotrophin receptor (p75NTR) is critical for neuronal survival and differentiation. However, the interaction between p75NTR and TrkA receptors required for this activity is not understood. Here, we report that a specific 29-amino acid peptide derived from the intracellular domain fragment of p75NTR interacts with and potentiates binding of NGF to TrkA-expressing cells, leading to increased neurite outgrowth in sympathetic neurons as a result of enhanced Erk1/2 and Akt signaling. An endogenous intracellular domain fragment of p75NTR (p75ICD) containing these 29 amino acids is produced by regulated proteolysis of the full-length receptor. We demonstrate that generation of this fragment is a requirement for p75NTR to facilitate TrkA signaling in neurons and propose that the juxtamembrane region of p75ICD acts to cause a conformational change within the extracellular domain of TrkA. This finding provides new insight into the mechanism by which p75NTR and TrkA interact to enhance neurotrophic signaling.

The Human G93A-Superoxide Dismutase-1 Mutation, Mitochondrial Glutathione and Apoptotic Cell Death

Mutations in Cu/Zn superoxide dismutase are a cause of motor neuron death in about 20% of cases of familial amyotrophic lateral sclerosis (ALS). Although the molecular mechanism of which these mutations induce motor neuron cell death is to a large extent unknown, there is significant evidence that effects on mitochondrial function and development of oxidative stress make a major contribution to the selective death of motor neurons in this disease. In this overview article we review the current understanding of mutant SOD1-mediated motor neuron degeneration in ALS with focus on oxidative damage and mitochondrial dysfunction. We also present novel information on the role of mitochondrial glutathione for the survival of NSC-34 cells stably transfected with the human SOD1G93A mutation, putting forward the hypothesis that this antioxidant pool provides a potentially useful target for therapeutic intervention.

The effects of transmembrane sequence and dimerization on cleavage of the p75 neurotrophin receptor by γ-secretase.

Background: p75 neurotrophin receptor (p75NTR) signaling is modulated by dimerization and regulated intramembrane proteolysis (RIP). Results: Transmembrane sequence and TrkA but not ligands regulate p75NTR homodimerization and γ-secretase cleavage. Conclusion: Although γ-secretase does not require a dimeric substrate, p75NTR dimerization facilitates RIP. Significance: Structural change mediated by homo- and heterodimerization is more important than ligand for inducing RIP of p75NTR. Cleavage of transmembrane receptors by γ-secretase is the final step in the process of regulated intramembrane proteolysis (RIP) and has a significant impact on receptor function. Although relatively little is known about the molecular mechanism of γ-secretase enzymatic activity, it is becoming clear that substrate dimerization and/or the α-helical structure of the substrate can regulate the site and rate of γ-secretase activity. Here we show that the transmembrane domain of the pan-neurotrophin receptor p75NTR, best known for regulating neuronal death, is sufficient for its homodimerization. Although the p75NTR ligands NGF and pro-NGF do not induce homerdimerization or RIP, homodimers of p75NTR are γ-secretase substrates. However, dimerization is not a requirement for p75NTR cleavage, suggesting that γ-secretase has the ability to recognize and cleave each receptor molecule independently. The transmembrane cysteine 257, which mediates covalent p75NTR interactions, is not crucial for homodimerization, but this residue is required for normal rates of γ-secretase cleavage. Similarly, mutation of the residues alanine 262 and glycine 266 of an AXXXG dimerization motif flanking the γ-secretase cleavage site within the p75NTR transmembrane domain alters the orientation of the domain and inhibits γ-secretase cleavage of p75NTR. Nonetheless, heteromer interactions of p75NTR with TrkA increase full-length p75NTR homodimerization, which in turn potentiates the rate of γ-cleavage following TrkA activation independently of rates of α-cleavage. These results provide support for the idea that the helical structure of the p75NTR transmembrane domain, which may be affected by co-receptor interactions, is a key element in γ-secretase-catalyzed cleavage.

Non-viral gene therapy that targets motor neurons in vivo

A major challenge in neurological gene therapy is safe delivery of transgenes to sufficient cell numbers from the circulation or periphery. This is particularly difficult for diseases involving spinal cord motor neurons such as amyotrophic lateral sclerosis (ALS). We have examined the feasibility of non-viral gene delivery to spinal motor neurons from intraperitoneal injections of plasmids carried by “immunogene” nanoparticles targeted for axonal retrograde transport using antibodies. PEGylated polyethylenimine (PEI-PEG12) as DNA carrier was conjugated to an antibody (MLR2) to the neurotrophin receptor p75 (p75NTR). We used a plasmid (pVIVO2) designed for in vivo gene delivery that produces minimal immune responses, has improved nuclear entry into post mitotic cells and also expresses green fluorescent protein (GFP). MLR2-PEI-PEG12 carried pVIVO2 and was specific for mouse motor neurons in mixed cultures containing astrocytes. While only 8% of motor neurons expressed GFP 72 h post transfection in vitro, when the immunogene was given intraperitonealy to neonatal C57BL/6J mice, GFP specific motor neuron expression was observed in 25.4% of lumbar, 18.3% of thoracic and 17.0% of cervical motor neurons, 72 h post transfection. PEI-PEG12 carrying pVIVO2 by itself did not transfect motor neurons in vivo, demonstrating the need for specificity via the p75NTR antibody MLR2. This is the first time that specific transfection of spinal motor neurons has been achieved from peripheral delivery of plasmid DNA as part of a non-viral gene delivery agent. These results stress the specificity and feasibility of immunogene delivery targeted for p75NTR expressing motor neurons, but suggests that further improvements are required to increase the transfection efficiency of motor neurons in vivo.

Inhibition of motor neuron death in vitro and in vivo by a p75 neurotrophin receptor intracellular domain fragment

ABSTRACT The p75 neurotrophin receptor (p75NTR; also known as NGFR) can mediate neuronal apoptosis in disease or following trauma, and facilitate survival through interactions with Trk receptors. Here we tested the ability of a p75NTR-derived trophic cell-permeable peptide, c29, to inhibit p75NTR-mediated motor neuron death. Acute c29 application to axotomized motor neuron axons decreased cell death, and systemic c29 treatment of SOD1G93A mice, a common model of amyotrophic lateral sclerosis, resulted in increased spinal motor neuron survival mid-disease as well as delayed disease onset. Coincident with this, c29 treatment of these mice reduced the production of p75NTR cleavage products. Although c29 treatment inhibited mature- and pro-nerve-growth-factor-induced death of cultured motor neurons, and these ligands induced the cleavage of p75NTR in motor-neuron-like NSC-34 cells, there was no direct effect of c29 on p75NTR cleavage. Rather, c29 promoted motor neuron survival in vitro by enhancing the activation of TrkB-dependent signaling pathways, provided that low levels of brain-derived neurotrophic factor (BDNF) were present, an effect that was replicated in vivo in SOD1G93A mice. We conclude that the c29 peptide facilitates BDNF-dependent survival of motor neurons in vitro and in vivo. Summary: A peptide mimetic of the p75NTR intracellular domain potentiates the survival of motor neurons in vivo by redressing the neurotrophin signaling imbalance in neurodegenerative conditions.