Mary-Louise Rogers

Antibody-Functionalized Porous Silicon Nanoparticles for Vectorization of Hydrophobic Drugs

We describe the preparation of biodegradable porous silicon nanoparticles (pSiNP) functionalized with cancer cell targeting antibodies and loaded with the hydrophobic anti-cancer drug camptothecin. Orientated immobilization of the antibody on the pSiNP is achieved using novel semicarbazide based bioconjugate chemistry. To demonstrate the generality of this targeting approach, the three antibodies MLR2, mAb528 and Rituximab are used, which target neuroblastoma, glioblastoma and B lymphoma cells, respectively. Successful targeting is demonstrated by means of flow cytometry and immunocytochemistry both with cell lines and primary cells. Cell viability assays after incubation with pSiNPs show selective killing of cells expressing the receptor corresponding to the antibody attached on the pSiNP.

Isolation and enrichment of embryonic mouse motoneurons from the lumbar spinal cord of individual mouse embryos

Cultured spinal motoneurons are a valuable tool for studying the basic mechanisms of axon and dendrite growth and also for analyses of pathomechanisms underlying diseases like amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). As motoneurons in the developing spinal cord of mice constitute only a minor population of neurons, these cells need to be enriched in order to study them in the absence of contaminating neuronal and non-neuronal cells. Here, we describe a protocol for the isolation and in vitro cultivation of embryonic primary motoneurons from individual mouse embryos. Tissue dissection, cell isolation and a p75NTR-antibody-based panning technique, which highly enriches motoneurons within <8 h are described. This protocol is aimed to provide an alternative to the established FACS-based protocols describing p75NTR-based enrichments of neurons. This protocol will help in facilitating the research on molecular mechanisms underlying motoneuron development, survival and disease mechanisms.

Non-viral gene therapy for neurological diseases, with an emphasis on targeted gene delivery.

Non-viral gene therapy systems are considered safer than viral delivery. This article reviews recent research describing novel, non-viral gene delivery to the central nervous system, with a special emphasis on receptor mediated gene delivery using antibodies (termed immunogenes) to specific receptors. By using targeting agents such as antibodies that can be retrogradely transported within neurons, non-viral gene therapies can deliver genes to specific neurons protected by the blood brain barrier. Components of effective non-viral gene therapy are described including DNA/RNA carriers, receptor-mediated endocytosis, endosomal escape and nuclear entry. In addition, stealth agents such as polyethylene glycol that can be used to improve in-vivo delivery are discussed. The value of immunogenes as therapeutic agents for fatal diseases such as Amyotrophic Lateral Sclerosis is significant but further in-vivo work to confirm efficacy is required before truly effective therapies can be achieved.

Milk-derived growth factors as serum supplements for the growth of fibroblast and epithelial cells

SummaryWe have investigated the response of several epithelial and fibroblastic cells to a mitogenic extract of bovine milk. Cation exchange chromatography was used to produce a mitogen-rich fraction from an industrial whey source that, although comprising only 0.5% of total whey protein, contained the bulk of the growth factor activity. This fraction was a source of potent growth promoting activity for all mesodermal-derived cells tested, including human skin and embryonic lung fibroblasts, Balb/c 3T3 fibroblasts, and rat L6 myoblasts. Maximal growth of all these cell types exceeded that observed in 10% fetal bovine serum. Feline kidney and baby hamster fibroblasts and Chinese hamster ovary cells were less responsive, achieving a maximal growth response of 50–75% that observed in 10% fetal bovine serum. Maximal growth achieved in whey-extract-supplemented cultures of Balb/c 3T3 and human skin fibroblasts, and L6 myoblast cultures exceeded that seen in response to recombinant acidic or basic fibroblast growth factor, platelet-derived growth factor, insulin-like growth factor, or epidermal growth factor. Importantly, addition of low concentrations of fetal bovine serum to the whey-derived mitogenic fraction produced an additive response. However, concentrated milk-derived factors were found to be inhibitory to the growth of all epithelial lines tested, including rat intestinal epithelial cells, canine kidney epithelial cells, and mink lung cells. It is concluded that industrial whey extracted in this form constitutes an important source of potent growth-promoting agents for the supplementation of mesodermal-derived cell cultures.

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.

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.

Identification of fibroblast growth factors in bovine cheese whey.

Acidic and basic fibroblast growth factors (FGF) were identified in bovine cheese whey after partial purification using a two step procedure. Cation-exchange chromatography produced a mitogen-rich extract which was loaded on to a heparin-sepharose column and eluted stepwise with 0.8, 1.2 and 2.0 M-NH4HCO3. Mitogenic activity was found in all three fractions by cell growth assays using Balb/c-3T3 fibroblasts. Immunoblotting identified acidic FGF in the 1.2 M-eluate and basic FGF in the 2.0 M-eluate, but neither acidic nor basic FGF was detected in the 0.8 M-fraction. Quantitative radioreceptor assays indicated 5.8 ng of acidic FGF-like activity and 19.8 ng of basic FGF-like activity per 1 whey in the appropriate eluates. This study represents the first direct demonstration of FGF in milk.

The Extracellular Domain of Neurotrophin Receptor p75 as a Candidate Biomarker for Amyotrophic Lateral Sclerosis

Objective biomarkers for amyotrophic lateral sclerosis would facilitate the discovery of new treatments. The common neurotrophin receptor p75 is up regulated and the extracellular domain cleaved from injured neurons and peripheral glia in amyotrophic lateral sclerosis. We have tested the hypothesis that urinary levels of extracellular neurotrophin receptor p75 serve as a biomarker for both human motor amyotrophic lateral sclerosis and the SOD1G93A mouse model of the disease. The extracellular domain of neurotrophin receptor p75 was identified in the urine of amyotrophic lateral sclerosis patients by an immuno-precipitation/western blot procedure and confirmed by mass spectrometry. An ELISA was established to measure urinary extracellular neurotrophin receptor p75. The mean value for urinary extracellular neurotrophin receptor p75 from 28 amyotrophic lateral sclerosis patients measured by ELISA was 7.9±0.5 ng/mg creatinine and this was significantly higher (p<0.001) than 12 controls (2.6±0.2 ng/mg creatinine) and 19 patients with other neurological disease (Parkinsons disease and Multiple Sclerosis; 4.1±0.2 ng/mg creatinine). Pilot data of disease progression rates in 14 MND patients indicates that p75NTRECD levels were significantly higher (p = 0.0041) in 7 rapidly progressing patients as compared to 7 with slowly progressing disease. Extracellular neurotrophin receptor p75 was also readily detected in SOD1G93A mice by immuno-precipitation/western blot before the onset of clinical symptoms. These findings indicate a significant relation between urinary extracellular neurotrophin receptor p75 levels and disease progression and suggests that it may be a useful marker of disease activity and progression in amyotrophic lateral sclerosis.