Mitchell Goldfarb - US grants

[unreadable] DESCRIPTION (provided by applicant): [unreadable] Fibroblast growth factor homologous factors (FHFs) bear sequence and structural homology to fibroblast growth factors (FGFs), but FHFs act solely as neuronal intracellular signalling molecules. FHFs are co-factors for the assembly of the kinase module and are themselves phosphorylated in vivo. FHFs have been shown to associate with a MAP kinase scaffold protein, IB2, and with voltage-gated sodium channels in the central and peripheral nervous systems. Although the precise functions of FHFs are still poorly understood, mice bearing mutations in FHF genes are neurologically impaired. FHF1-/-FHF4 -/- double knockout mice are severely hyperactive and have very poor grip strength. Preliminary electromyography data indicate motor nerve and neuromuscular deficiencies in these animals. Continued research on FHFs shall focus on four new Specific Aims: I) The nerve conduction and neuromuscular transmission defects of FHF1-/-FHF4 -/- mice shall be determined through electrophysiological, ultrastructural, immunological, and biochemical approaches. Detected motor nerve defects shall guide analysis of higher brain centers for similar defects. Genetic interaction between mutant FHF and sodium channel genes shall be investigated. II) The IB2 gene shall be disrupted conditionally in neurons of neonatal mice. The motor phenotypes of IB2 mutant mice shall be determined and compared to those of FHF mutants, and we shall test whether reduced IB2 gene dosage potentiates FHF mutant phenotypes. III) The identity and constituents of brain microvesicles with which phosphorylated FHF associate shall be determined by conventional and affinity-based fractionation methods. IV) The structure of complexes containing FHF together with the FHF-binding segment of IB2 or sodium channels shall be determined by X-ray crystallographic methods. The structures shall guide mutagenesis to determine residues critical for interactions, determine whether IB2 and sodium channels bind FHF by a similar mechanism, and resolve why FHF and FGF folds and surfaces are so paradoxically similar. [unreadable] [unreadable]

Fibroblast growth factor homologous factors (FHFs) are intracellular neuromodulators whose mechanisms of action are still poorly understood. Mice bearing deletions in Fhf genes display neurological deficits associated with motor[unreadable] function, although the animals show no detectable histological or immunohistochemical abnormalities. FHFs are[unreadable] believed to exert their effects through identified binding partners, which include voltage-gated sodium channels.[unreadable] Recent findings from the P.I.'s lab show that FHFs are required for proper intrinsic excitability of neurons, and that[unreadable] FHF-deficient neurons show aberrant voltage-dependent sodium channel behavior. Further data show that FHFs are[unreadable] required for optimal conduction of action potentials along motor axons. In light of these findings, this application[unreadable] proposes experiments to answer four related questions concerning FHF physiology:[unreadable] I. Do FHFs modulate neuronal excitability through direct FHF-sodium channel interactions?[unreadable] II. Are different FHF genes and protein isoforms equivalent or distinct in terms of sodium channel modulation and[unreadable] excitability?[unreadable] III. Does temporal regulation of FHF protein levels contribute to plasticity of intrinsic excitability?[unreadable] IV. Do FHF protein levels impact on the functional severity of demyelination syndromes?[unreadable] These experiments are expected to more fully define the mechanisms and conditions by which FHFs control physiology[unreadable] and pathophysiology of the central nervous system. More specifically, these studies will analyze FHF genes as[unreadable] potential contributors to severity of Charcot-Marie-Tooth syndrome and diabetic peripheral neuropathy, the latter of[unreadable] which constitutes a significant minority health disparity.

DESCRIPTION (provided by applicant): Voltage-gated sodium channels (VGSCs) generate and propagate electrical activity in excitable cells. The precise dynamics of VGSC transitions among closed, open, and inactivated states are essential for brain, nerve, heart, and muscle function, as documented by a wide range of clinical sodium channelopathy disorders. While the VGSC alpha subunit in isolation comprises the channel's pore, its voltage sensors, and its inactivation machinery, interaction with fibroblast growth factor homologous factors (FHFs) has large and complex effects on the dynamics of VGSC inactivation. Indeed, FHF mutations are a cause of human spinocerebellar ataxia and cause defects in neuronal intrinsic excitability. While all FHF isoforms bind VGSCs, they differ in their abilities to modulate VGSC fast inactivation and to induce a newly characterized long-term inactivated channel state. This application proposes experiments to expand analysis of FHF neuronal functions and physical mechanisms of FHF- induced VGSC modulation. AIM I: Our overall biological hypothesis is that differential expression of FHF isoforms in different neurons and their subcellular compartments acts to determine excitation and conduction properties of cells. We will test: (I-A) Does the relative abundance of different long-form FHFs (A-type FHFs, FHF4B) at the axon initial segment specify the excitation properties of a neuron? (I-B) Does somatodendritic membrane localization of A-type FHFs act to limit sodium action potential backpropagation during repetitive firing of neurons? (I-C) Are long-form FHFs not required for, and potentially deleterious to, action potential axonal conduction? AIM II: A clearer understanding of physical mechanisms for VGSC modulation by FHFs will provide further insight into VGSC conformation dynamics and suggest rational approaches for designing therapeutics for managing disorders of hyperexcitability, such as epilepsies and arrhythmias. We will test: (II-A) Does the long- term inactivation particle at the distal N-terminus of A-type FHFs dock within the cytoplasmic cavern of a VGSC? (II-B) Do the cationic residues in the docked A-type FHF inactivation particle act as a shield against sodium ion conduction? (II-C) How does a cationic region near the ?-trefoil core of FHFs modulate VGSC fast inactivation? PUBLIC HEALTH RELEVANCE: Voltage-gated sodium channels (VGSCs) generate and propagate electrical activity in excitable cells. The speed by which VGSCs shuttle among closed, open, and inactive states is modulated by a family of proteins called FHFs. We propose experiments to further determine the roles of FHFs in the control of nerve cell electrical activity and to determine the physical mechanisms exploited by FHFs to modulate VGSC conformational dynamics.

DESCRIPTION (provided by applicant): One of the major obstacles to axonal regeneration in the adult CNS is inhibitors associated with myelin, such as MAG. However, axonal growth can be encouraged in an inhibitory environment both in culture and in vivo if the neuronal cAMP levels are elevated, either with analogues such as db cAMP or by priming neurons with neurotrophins. One situation where spontaneous CNS axon regeneration does occur is of dorsal root ganglion axons if the peripheral branch of the same neuron, the dorsal root ganglion (DRG) neuron, is lesioned one week before - the conditioning lesion (CL) effect, which is cAMP dependent. Both the cAMP and the CL effects are dependent on transcription and one gen that is up-regulated is for the enzyme Arginase I (Arg I), which is key in the synthesis of polyamines. The polyamine, spermidine, can overcome inhibition by MAG in culture and promotes optic nerve regeneration in vivo. Furthermore, spermidine promotes regeneration by activating the kinase CDK5 by hypusinating the eukaryotic initiation factor 5A (eIF5A), resulting in an increase in translation of the CDK5 activator p35. In Aim 1a the CDK5 substrates that are activated in response to polyamine will be identified and characterized for their role in overcoming inhibition and promoting regeneration in vivo. Aim 1b will address the cross-talk between the neurotrophin and MAG signaling pathway, focusing on the ability of MAG to block the activation of the small GTPase, Rap1, by neurotrophin. Strong preliminary data suggest that both the cAMP and CL effects require local translation in the axon to promote regeneration in an inhibitory environment. In Aim 2 mRNA and microRNAs that increase in the processes after both a CL and treatment with db cAMP will be identified. In Aim 3 those mRNAs and microRNAs that increase after both conditions will be characterized (over-expression, knock-down) for a possible role in promoting regeneration in the presence of MAG in culture and in promoting regeneration in vivo. Through the experiments described in this proposal not only will our understanding be advanced of the mechanism of action of agents known to promote regeneration in vivo but novel agents will be identified. This in turn will reveal novel targets for intervention and drug development to promote axonal regeneration in vivo.