Nicola Neff

Insulin-like Growth Factor-I: Potential for Treatment of Motor Neuronal Disorders

Motor neuronal disorders, such as the loss of spinal cord motor neurons in amyotrophic lateral sclerosis or the degeneration of spinal cord motor neuron axons in certain peripheral neuropathies, present a unique opportunity for therapeutic intervention with neurotrophic proteins. Normally, such proteins do not cross the blood-brain barrier, but spinal cord motor neuron axons and nerve terminals lie outside the barrier and thus may be targeted by systemic administration of protein growth factors. Insulin-like growth factor-I (IGF-I) receptors are present in the spinal cord, and, like members of the neurotrophin receptor family, IGF-I receptors mediate signal transduction via a tyrosine kinase domain. IGF-I was found to prevent the loss of choline acetyltransferase activity in embryonic spinal cord cultures, as well as to reduce the programmed cell death of motor neurons in vivo during normal development or following axotomy or spinal transection. Consistent with earlier reports that IGF-I enhances motor neuronal sprouting in vivo, subcutaneous administration of IGF-I increases muscle endplate size in rats. Subcutaneous injections of IGF-I also accelerate functional recovery following sciatic nerve crush in mice, as well as attenuate the peripheral motor neuropathy induced by chronic administration of the cancer chemotherapeutic agent vincristine in mice. Doses of IGF-I that accelerate recovery from sciatic nerve crush in mice result in elevated serum levels of IGF-I which are similar to those obtained following subcutaneous injections of formulated recombinant human IGF-I (Myotrophin) in normal human subjects. Based on these findings, together with evidence of safety in animals and man, clinical trials of recombinant human IGF-I have been initiated in patients with amyotrophic lateral sclerosis and are planned to begin soon in patients with chemotherapy-induced peripheral neuropathies.

CEP‐751 inhibits trk receptor tyrosine kinase activity in vitro and exhibits anti‐tumor activity

The present report describes the in vitro and in vivo profile of CEP‐751, a novel receptor tyrosine kinase inhibitor. CEP‐751 at 100 nM inhibits the receptor tyrosine kinase activity of the neurotrophin receptors trkA, trkB and trkC. CEP‐751 has no effect on activity of receptors for EGF, IGF‐I, insulin or on erbB2; inhibition of receptors for PDGF and bFGF was observed but occurred with lesser potency than inhibition of trk. CEP‐751 exhibited anti‐tumor efficacy against tumors derived from NIH3T3 cells transfected with trkA. Inhibition of trk phosphorylation could also be measured in these tumors, suggesting that anti‐tumor efficacy of CEP‐751 is related to inhibition of trk receptor tyrosine kinase activity. CEP‐751 was found to be without effect when administered to nude mice bearing SK‐OV‐3 tumors, which overexpress erbB2 receptors, providing further evidence that inhibition of tumor growth may be related to inhibition of trk receptor tyrosine kinase activity. Our data indicate that CEP‐751 is a potent trk inhibitor which possesses anti‐tumor activity. Int. J. Cancer 72:673–679, 1997.

CEP-1347/KT7515 prevents motor neuronal programmed cell death and injury-induced dedifferentiation in vivo.

CEP-1347, also known as KT7515, a derivative of a natural product indolocarbazole, inhibited motor neuronal death in vitro, inhibited activation of the stress-activated kinase JNK1 (c-jun NH terminal kinase) in cultured spinal motor neurons, but had no effect on the mitogen-activated protein kinase ERK1 in these cells. Results reported here profile the functional activity of CEP-1347/KT7515 in vivo in models of motor neuronal death or dedifferentiation. Application of CEP-1347/KT7515 to the chorioallantoic membrane of embryonic chicks rescued 40% of the lumbar motor neurons that normally die during the developmental period assessed. Peripheral administration of low doses (0.5 and 1 mg/kg daily) of CEP-1347/KT7515 reduced death of motor neurons of the spinal nucleus of the bulbocavernosus in postnatal female rats, with efficacy comparable to testosterone. Strikingly, daily administration of CEP-1347/KT7515 during the 4-day postnatal window of motor neuronal death resulted in persistent long-term motor neuronal survival in adult animals that received no additional CEP-1347/KT7515. In a model of adult motor neuronal dedifferentiation following axotomy, local application of CEP-1347/KT7515 to the transected hypoglossal nerve substantially reduced the loss of choline acetyl transferase immunoreactivity observed 7 days postaxotomy compared to untreated animals. Results from these experiments demonstrate that a small organic molecule that inhibits a signaling pathway associated with stress and injury also reduces neuronal death and degeneration in vivo.

A rapid fluorometric assay to measure neuronal survival in vitro.

We report the development and characterization of a rapid fluorometric microassay suitable for quantifying neuronal cell survival. The method can be used in two formats: (1) a time course analysis of survival response or (2) as a simple endpoint assay for the assessment of neuronal survival promoted by a variety of reagents. The assay uses calcein AM, a non-fluorescent, electrically neutral, non-polar analogue of fluorescein diacetate, which passively crosses cell membranes and is cleaved to a fluorescent derivative by non-specific intracellular esterases. Once cleaved in viable cells, the resultant fluorescent salts are retained by intact cell membranes. The relative number of viable cells under various conditions can be quantified by measuring the emitted fluorescence. Described herein are the conditions that allow the determination of low viable neuronal cell numbers (10(2)-10(3) cells/cm2).

K‐252a Induces Tyrosine Phosphorylation of the Focal Adhesion Kinase and Neurite Outgrowth in Human Neuroblastoma SH‐SY5Y Cells

Abstract: The protein kinase inhibitor K‐252a has been shown to promote cholinergic activity in cultures of rat spinal cord and neuronal survival in chick dorsal root ganglion cultures. To determine the mechanism by which K‐252a acts as a neurotrophic factor, we examined the effects of this molecule on a human neuroblastoma cell line, SH‐SY5Y. K‐252a induced neurite outgrowth in a dose‐dependent manner. Coincident with neurite outgrowth was the early tyrosine phosphorylation of 125‐ and 140‐kDa proteins. The phosphorylation events were independent of protein kinase C inhibition because down‐regulation of protein kinase C by long‐term treatment with phorbol ester did not prevent K‐252a‐induced tyrosine phosphorylation. Similarly, the protein kinase C inhibitors H7, GF‐109203X, and calphostin C did not induce the phosphorylation. We have identified one of the phosphosubstrates as the pp125 focal adhesion protein tyrosine kinase (Fak). Induction of phosphorylation coincided with increased Fak activity and appeared to be independent of ligand/integrin interaction. The induction of Fak phosphorylation by K‐252a was also observed in LA‐N‐5 cells and primary cultures of rat embryonic striatal cells but not in PC12 cells. The protein kinase C‐independent induction of tyrosine phosphorylation and the identification of Fak as a substrate of K‐252a‐induced tyrosine kinase activity suggest that this compound mediates neurotrophic effects through a novel signaling pathway.

K-252a and Staurosporine Promote Choline Acetyltransferase Activity in Rat Spinal Cord Cultures

Abstract: The protein kinase inhibitor K‐252a increased choline acetyltransferase (ChAT) activity in rat embryonic spinal cord cultures in a dose‐dependent manner (EC50 of ∼100 nM) with maximal stimulatory activity at 300 nM resulting in as much as a fourfold increase. A single application of K‐252a completely prevented the marked decline in ChAT activity occurring over a 5‐day period following culture initiation. Of 11 kinase inhibitors, only the structurally related inhibitor Staurosporine also increased ChAT activity (EC50 of ∼0.5 nM). Effective concentrations of K‐252a were not cytotoxic or mitogenic and did not alter the total protein content of treated cultures. Insulin‐like growth factor I, basic fibroblast growth factor, ciliary neurotrophic factor, and leukemia inhibitory factor yielded dose‐dependent increases in ChAT activity in spinal cord cultures. The combination of K‐252a with insulin‐like growth factor‐l or basic fibroblast growth factor increased ChAT activity up to eightfold over that of untreated controls, which was greater than that observed with each compound alone. K‐252a combined with ciliary neurotrophic factor or leukemia inhibitory factor demonstrated no additive or synergistic effects on ChAT activity. These results suggest that there are multiple mechanisms for the regulation of ChAT activity in spinal cord cultures. The enhancement of spinal cord ChAT activity by K‐252a and Staurosporine defines a new neurotrophic activity for these small organic molecules and raises the possibility that they may activate some regulatory elements in common with the ciliary neurotrophic factor and leukemia inhibitory factor family of neurotrophic proteins.

K‐252a Promotes Survival and Choline Acetyltransferase Activity in Striatal and Basal Forebrain Neuronal Cultures

Abstract: The organic molecule K‐252a promoted cell survival, neurite outgrowth, and increased choline acetyltransferase (ChAT) activity in rat embryonic striatal and basal forebrain cultures in a concentration‐dependent manner. A two‐ to threefold increase in survival was observed at 75 nM K‐252a in both systems. A single application of K‐252a at culture initiation prevented substantial (>60%) cell death that otherwise occurred after 4 days in striatal or basal forebrain cultures. A 5‐h exposure of striatal or basal forebrain cells to K‐252a, followed by its removal, resulted in survival equivalent to that observed in cultures continually maintained in its presence. This is in contrast to results found with a 5‐h exposure of basal forebrain cultures to nerve growth factor (NGF). Acute exposure of basal forebrain cultures to K‐252a, but not to NGF, increased ChAT activity, indicating that NGF was required the entire culture period for maximum activity. Striatal cholinergic and GABAergic neurons were among the neurons rescued by K‐252a. Of the protein growth factors tested in striatal cultures (ciliary neurotrophic factor, neurotrophin‐3, NGF, brain‐derived neurotrophic factor, interleukin‐2, basic fibroblast growth factor), only brain‐derived neurotrophic factor promoted survival. The enhancement of survival and ChAT activity of basal forebrain and striatal neurons by K‐252a defines additional populations of neurons in which survival and/or differentiation is regulated by a K‐252a‐responsive mechanism. The above results expand the potential therapeutic targets for these molecules for the treatment of neurodegenerative diseases.

CEP-1347/KT7515, a JNK pathway inhibitor, supports the in vitro survival of chick embryonic neurons.

DEVELOPING neurons depend on target-derived trophic factors for survival in vivo and in vitro, which also decrease the activity of c-Jun N-terminal kinase (JNK). We have recently described a survival-promoting effect of inhibitors of cyclin-dependent kinases and JNK on chick peripheral embryonic neurons. Here, we report that the small trophic molecule CEP-1347/KT7515, which has been shown to inhibit the JNK signalling pathway, can promote long term-survival of cultured chick embryonic dorsal root ganglion, sympathetic, ciliary and motor neurons. Because of their pharmacological properties, small trophic molecules such as CEP-1347/KT7515 might be of interest for the treatment of neurodegenerative disorders.

The effect of Pyrrolo[3,4-c]carbazole derivatives on spinal cord ChAT activity

Abstract Pyrrolo[3,4-c]carbazole derivatives were prepared as potential neurotrophic agents. The compounds were assayed for their ability to stimulate choline acetyltransferase (ChAT) activity in embryonic rat spinal cord cultures. These simplified K252a derivatives, although less potent and efficacious, have led to the identification of minimal structural requirements for K252a neurotrophic activity.

AN ELISA ASSAY FOR GAP-43

An ELISA assay for the growth associated protein GAP-43 was developed to determine rapidly its relative abundance in neuronal tissue. The assay was performed with affinity-purified anti-GAP-43 antibody that detected a single band of Mr = 42,000-45,000 on Western blots of rat brain homogenates but no bands on blots of liver homogenates. GAP-43 was determined by ELISA assay in as little as 0.6 microgram protein of brain homogenate. The assay was highly reproducible; the standard error of the mean of sample to sample variation was less than 5%. When ELISA development time was held constant, the standard error of the mean of inter-assay variation was between 2 and 7%. Using this method, GAP-43 immunoreactivity was examined in developing rat brain. At post-natal day 1, GAP-43 immunoreactivity was 3-4 times greater than that observed in the adult, remained elevated for several weeks, and decreased by the end of the first month of life. These results are in accord with previous studies on the expression or synthesis of GAP-43 during neuronal development.