Douglas Ishii - US grants

Our objective is to study the effects of insulin and insulin-like growth factor II (IGF-II), or lack thereof, in the development and growth of the sensory and sympathetic nervous system, in malignant human neuroblastoma cells, and in diabetes. Diabetic neuropathy is characterized in part by reduced conduction velocity, reduced nerve fiber diameter and loss of axons. Human neuroblastoma is characterized in part by escape from the requirement of nerve growth factor for survival and for growth regulation (K.H. Sonnenfeld and D.N. Ishii, J. Neurosci. Res. in press, 1982). One means by which escape may occur is through acquisition of inappropriate sensitivity to other growth factors. Our recent findings show that neuroblastoma cells are stimulated to divide by insulin and IGF-II, but not late stage embryonic sensory and sympathetic neurons in culture. Whether earlier developmental stages are affected is not known. Moreover, insulin can enhance neurite outgrowth in neuroblastoma cells. These results suggest insulin may have direct effects on the peripheral nervous system, and whether diabetic neuropathy is the consequence of direct or indirect insulin effects may be tested. Our aims are: a) to more completely study how insulin and IGF-II affects growth, survival and neurite outgrowth in neuroblastoma and in pheochromocytoma PC12 cells, b) to determine whether neuroblastoma cells can synthesize insulin and produce its own growth factor, c) to study whether insulin can alter response to nerve growth factor, d) to study the role of insulin, IGF-II, and anti-insulin serum on maturation, growth, protein synthesis, and survival of sensory and sympathetic neuroblasts during development, and e) to study the time course and magnitude of effects on nerve growth factor binding, axonal transport, protein synthesis, survival, and specific enzyme activity following onset of diabetes in rats to test whether insulin may directly be involved in neuropathy, and explain the syndrome described above.

The long term objective is to study insulinlike growth factor (IGF) and insulin's neurophysiological role, mechanism for regulation of neurite growth, and involvement in neuronal disorders. In this Project period, the following will be studied. A) The mechanism of neurite formation is a fundamental neurobiological problem whose solution may permit manipulation of axon regeneration. We will determine whether tubulin mRNA levels are commonly regulated by neuritogenic polypeptides as a prelude to neurite formation in cultured sensory, sympathetic, and PC12 cells. B) We will study the developmental and tissue specific regulation of IGF-II gene expression, and the relationship between gene expression and innervation in the rat. The neuritogenic potential of insulin and IGF-I in spinal cord cultures will be examined. Following the intrathecal administration of insulin, anti-insulin- antiserum, and anti-IGF-I antiserum, spinal cord will be tested for changes in conduction velocity, microscopic anatomy, and levels of NGF, IGF-I, and tubulin mRNAs. C) Neuropathy is a common complication of diabetes, a disorder affecting millions of individuals. The National Diabetes Advisory Board recommend the need to better understand its fundamental pathophysiology, and sequence of pathophysiologic events, so that the hypotheses concerned with its causation may be both newly formulated and tested to the end that better modalities of treatment may ultimately be found. Despite the microscopic lesions found in human spinal cord in post-mortem studies, convincing evidence of functional pathology has been difficult to obtain because virtually all the signs and symptoms of spinal cord involvement are masked by the peripheral neuropathy. Following the induction of diabetes with streptozotocin in the rat, we shall correlate the temporal sequence for the emergence of microscopic lesions with alterations in spinal cord conduction velocity, and levels of IGF-I, NGF, and tubulins mRNAs. Whether these alterations can be ameliorated by intrathecal insulin administration will be evaluated. These studies will simultaneously test our novel hypothesis for the pathogenesis of diabetic neuropathy. Mice will be used to isolate NGF, rabbits to produce antisera, and fertile eggs for neurite studies.

The primary purpose of this study is to investigate the role and mechanism of insulin like growth factor-II (IGF-II) in the nervous system. The identification of new neuritogenic factors, and the determination of their mechanisms, would significantly advance our understanding of nervous system development. IGF-II is a member of a new family of related neuritogenic polypeptides. Transcripts related to the pre-pro-IGF-II mRNA sequence are expressed in brain and other tissues. We propose, in this project period, to study more completely the expression of the IGF-II gene in various rat tissues during development, and following denervation. These studies will determine whether the temporal expression of IGF-II transcripts is consistent with innervation and maturation of target tissues, and reveal the intimate relationship between gene expression and innervation. The requirement of IGF-II for development of the sensory and sympathetic nervous system will be studied by treatment of neonatal rats with anti- IGF-II antiserum. The mechanism by which IGF-II enhances neurite formation will be studied in cultured sensory and sympathetic neurons from chick and rat, and in human neuroblastoma and rat pheochromocytoma cell lines. The mechanism of neurite outgrowth, in itself, is a major, incompletely solved problems, whose solution could profoundly influence the fundamental understanding of neurobiology, and impact on significant clinical issues. The levels of tubulin mRNA appear to be highly regulated by IGF-II, insulin and nerve growth factor in a variety of cell types, and correlate with neurite formation. We shall study whether tubulin mRNA expression is essential for neurite growth, document more completely its regulation by these factors, and study the mechanism for enhancement of tubulin mRNA levels. These studies serve to bridge the gap between neurite growth and attempts to identify the second messenger of neuritogenic polypeptides. Mice will be employed in this study as a source of nerve growth factor.

The primary purpose of this study is to investigate the role and mechanism of insulin like growth factor-II (IGF-II) in the nervous system. The identification of new neuritogenic factors, and the determination of their mechanisms, would significantly advance our understanding of nervous system development. IGF-II is a member of a new family of related neuritogenic polypeptides. Transcripts related to the pre-pro-IGF-II mRNA sequence are expressed in brain and other tissues. We propose, in this project period, to study more completely the expression of the IGF-II gene in various rat tissues during development, and following denervation. These studies will determine whether the temporal expression of IGF-II transcripts is consistent with innervation and maturation of target tissues, and reveal the intimate relationship between gene expression and innervation. The requirement of IGF-II for development of the sensory and sympathetic nervous system will be studied by treatment of neonatal rats with anti- IGF-II antiserum. The mechanism by which IGF-II enhances neurite formation will be studied in cultured sensory and sympathetic neurons from chick and rat, and in human neuroblastoma and rat pheochromocytoma cell lines. The mechanism of neurite outgrowth, in itself, is a major, incompletely solved problems, whose solution could profoundly influence the fundamental understanding of neurobiology, and impact on significant clinical issues. The levels of tubulin mRNA appear to be highly regulated by IGF-II, insulin and nerve growth factor in a variety of cell types, and correlate with neurite formation. We shall study whether tubulin mRNA expression is essential for neurite growth, document more completely its regulation by these factors, and study the mechanism for enhancement of tubulin mRNA levels. These studies serve to bridge the gap between neurite growth and attempts to identify the second messenger of neuritogenic polypeptides. Mice will be employed in this study as a source of nerve growth factor.

The incidence of diabetes is rising throughout the world due to increased industrialization, and nationally with the "greying" of America. Neuropathy is a highly debilitating consequence of diabetes in about 10% of patients. Particularly distressing problems include third nerve palsy, muscular weakness, bowel dysfunction, bladder dysfunction, impotence, heart rate abnormalities, severe unremitting pain, diabetic foot, and limb amputation. The economic costs to the individual and society are considerable, and there is a pressing need for new modalities of treatment, as well as a better understanding of the underlying cause of neuropathy. These are among recommendations of the National Diabetes Advisory Board. The long-term goal of this project is to study the medically relevant neurobiology of insulin-like growth factors (IGFs). This renewal application proposes continued study of two highly promising, independent but interrelated hypotheses: (i) Exogenously administered IGFs can prevent or ameliorate the syndrome of diabetic neuropathy, and (ii) IGFs play a role in the pathogenesis of diabetic neuropathy. A new theory for pathogenesis is proposed, and preliminary tests of key predictions of the theory have been validated. In a striking observation, it was found that locally infused IGF-II can prevent impairment of conduction velocity in diabetic rats, despite continued hyperglycemia. The specific aims are to determine whether infused IGFs can prevent and/or reverse impairment of a range of neural functions known to be afflicted in diabetic rats, including conduction velocity, nerve regeneration, size spectra of neuronal cell bodies, and functional regeneration. Moreover, experiments will determine whether endogenous IGF mRNA content is reduced in afflicted nerve and nerve targets in diabetic rats. The results may directly lead to new therapies for treatment of diabetic neuropathy. The main studies will be done with rats, mice will provide NGF, and fertile eggs will be used to bioassay IGFs and NGF.