Susan Carswell

Chronic 1,25-dihydroxyvitamin D3-mediated induction of nerve growth factor mRNA and protein in L929 fibroblasts and in adult rat brain

We have proposed that elevating levels of nerve growth factor (NGF) in the CNS is a rational strategy for treating certain neurodegenerative disorders. The present studies were conducted to determine: (1) if pharmacologically induced levels of NGF could be sustained for an extended time, and (2) if correlations exist between increases in NGF mRNA and NGF protein in L929 cells and in vivo. Short-term treatment of L929 cells with 1,25-dihydroxyvitamin D3 resulted in a two-fold increase in both NGF mRNA and NGF protein. These increases were sustained for up to 48 h with continuous exposure to 1,25-dihydroxyvitamin D3. In rats, 1,25-dihydroxyvitamin D3 (2.5 nmol; i.c.v.) induced NGF mRNA transiently, with peak two-fold increases observed 4 h post-injection. In contrast to L929 cells, 1,25-dihydroxyvitamin D3 did not elicit an increase in NGF protein after a single administration in vivo. However, consistent with long-term exposure in L929 cells, chronic 6 day infusion of 1,25-dihydroxyvitamin D3 resulted in induction of both NGF mRNA and NGF protein in the brain. These results indicate that 1,25-dihydroxyvitamin D3-mediated NGF induction in cultured L929 cells may predict of NGF induction in vivo, suggesting that L929 cells may have utility in studying underlying mechanisms of NGF induction by 1,25-dihydroxyvitamin D3. On the basis of NGFs ability to increase cholinergic function in animal models of cholinergic degeneration, these results are supportive of a role for NGF inducers as potential drugs for neurodegenerative disorders.

Pharmacological Induction of Nerve Growth Factor mRNA in Adult Rat Brain

Three structurally unrelated compounds, all of which induce nerve growth factor (NGF) in cell culture systems, were assessed for their ability to induce NGF mRNA in adult rat brain using a highly sensitive RNAse protection assay. Interleukin-1 beta (0.5-1 pmol) and 1,25-dihydroxyvitamin D3 (25-25,000 pmol) were extremely potent inducers of NGF mRNA, being respectively at least 50,000 and 4000 times more potent than 4-methylcatechol. These compounds elicited an approximate twofold increase in NGF mRNA in both the hippocampus and cortex, without altering beta-actin mRNA levels after a single intracerebroventricular injection. The duration of NGF induction was dependent on the compound administered. For example, the elevation of NGF mRNA elicited by interleukin-1 beta peaked at 8 h and lasted for at least 24 h. In contrast, the induction of NGF after 1,25-dihydroxyvitamin D3 and 4-methylcatechol administration peaked between 4 and 8 h and was not apparent 24 h after injection. These results demonstrate induction of NGF mRNA in vivo by administration of physiological or pharmacological agents and differentiate these agents by potency and duration of action. Further, these findings indicate that pharmacological induction of NGF may be a viable strategy for the treatment of neurodegenerative disorders such as Alzheimers disease.

1α, 25 dihydroxyvitamin D3-dependent up-regulation of calcium-binding proteins in motoneuron cells

Our understanding of selective neuronal vulnerability as well as etiopathogenesis of sporadic neurodegenerative diseases is extremely limited. In ALS, altered calcium homeostasis appears to contribute significantly to selective neuronal injury. Further in ALS, the absence of calcium binding proteins (calbindin‐D28K, parvalbumin, and calretinin) correlates with selective vulnerability and cell loss. In motoneuron cell culture models an ALS IgG‐triggered and calcium‐mediated destruction can be reversed by increased expression of calbindin‐D28K following retroviral infection with calbindin‐D28K cDNA. To increase calcium binding protein expression in motoneurons in vitro and in vivo, we have employed vitamin D3. Forty‐eight hr treatment of differentiated VSC 4.1 cells with 0.1–30 nM 1,25 dihydroxyvitamin D3 induced a twofold increase in the immunoreactivity for calbindin‐D28K and parvalbumin. Injection of 80–120 ng, 1,25 dihydroxyvitamin D3 in the cerebral ventricles of adult rats also induced positive immunoreactivity for calcium binding proteins in ventral motoneurons which are completely devoid of such reactivity in the adult stage. These data suggest that analogs of 1,25 dihydroxyvitamin D3 may be useful tools in enhancing the expression of calcium binding proteins in the motor system and may have possible therapeutic value in neurodegenerative disease.

Preservation of cholinergic activity and prevention of neuron death by CEP-1347/KT-7515 following excitotoxic injury of the nucleus basalis magnocellularis

We have identified a class of small organic molecules, derived from the indolocarbazole K-252a, that promote the survival of cultured neurons. However, many of these indolocarbazoles inhibit protein kinase C and neurotrophin-activated tyrosine kinase receptors. These kinase inhibitory activities may limit the utility of these compounds for neurological disorders. A bis-ethyl-thiomethyl analogue of K-252a, CEP-1347/KT-7515, has been identified that lacks protein kinase C and tyrosine kinase receptor inhibitory activities, yet retains the ability to promote survival of cultured neurons, including cholinergic neurons derived from the basal forebrain. In the present studies, CEP-1347/KT-7515 was assessed for neurotrophic activity on basal forebrain neurons of in vivo rats following excitotoxic insult. Ibotenate infusion into the nucleus basalis magnocellularis reduced levels of choline acetyltransferase activity in the cortex, as well as reduced numbers of choline acetyltransferase-immunoreactive and retrogradely (FluoroGold)-labelled cortically-projecting neurons in the nucleus basalis. Systemically administered CEP-1347/KT-7515 attenuated the loss of cortical choline acetyltransferase activity and the loss of the number of choline acetyltransferase-immunoreactive and retrogradely-labelled FluoroGold neurons in the nucleus basalis. Moreover, CEP-1347/KT-7515 ameliorated the loss of cortical choline acetyltransferase if administration was initiated one day, but not seven days post-lesion. Together, these results demonstrate that CEP-1347/KT-7515 protects damaged cortically-projecting basal forebrain neurons from degeneration. Thus, CEP-1347/KT-7515 may have therapeutic potential in neurodegenerative diseases, such as Alzheimers disease, in which basal forebrain cholinergic neurons degenerate.

Induction of NGF by isoproterenol, 4-methylcatechol and serum occurs by three distinct mechanisms

Evidence is provided that isoproterenol, 4-methylcatechol and serum induce NGF by three separate mechanisms. Isoproterenol and 4-methylcatechol induced NGF and NGF mRNA in mouse fibroblast L929 cells in either the presence or absence of serum. Propranolol prevented NGF induction by isoproterenol, but not by 4-methylcatechol or serum. All possible combinations of these inducers resulted in additive increases in the levels of NGF and NGF mRNA.

Therapeutic potential of nerve growth factor in Alzheimer's disease

Abstract The loss of basal forebrain cholinergic neurons is thought to be an important contributing factor in the cognitive impairments associated with Alzheimers disease. Based on its efficacy in several in vivo models of basal forebrain cholinergic neuronal degeneration in adult animals, nerve growth factor (NGF) is being considered as a therapeutic candidate for the treatment of this disorder. In rats and monkeys with partial transections or aspirations of the septo-hippocampal pathway, also called fimbria fornix lesions, the cell bodies of lesioned cholinergic cells atrophy and/or die as measured by loss of phenotypic markers. Markers of functional axonal terminals of these cells are also lost. The lesion also results in impaired performance in behavioral tasks of learning and memory. When exogenous NGF is supplied to the brain, however, cell body loss is almost fully prevented. Although NGF does not elicit reconnection of severed axons, it does enhance sprouting of intact neurons and increases expression of hippocampal cholinergic neuronal terminal markers. These increases in the expression of neuronal markers are accompanied by functional recovery, as measured in cognitive behavioral tasks. Analogous results occur in experimental animals with excitotoxic lesions of a distinctive group of basal forebrain cholinergic neurons, those of the nucleus basalis magnocellularis (NBM). These are a diffuse set of cells that project to the frontal-parietal cortex. The lesion produces neurochemical losses in terminal field markers and deficits in behavioral performance, which exogenously supplied NGF largely prevents. Rats also show a spontaneous age-associated decline in cognition, as measured in tasks of memory and learning, a component of which is thought to be ascribable to the loss of cholinergic cell function. Again, NGF improves performance in these animals. Together, this data provides a compelling rationale for the use of NGF in Alzheimers disease to slow the progression of cholinergic cell loss and the associated decline in cognition. The major impediment to the development of NGF for use in the human disease is its inability to cross the blood-brain barrier. Alternative approaches that exploit or mimic the beneficial effects of NGF for cholinergic neurons of the basal forebrain while overcoming the delivery problems are also examined.