The role of human endogenous retroviruses (HERVs) in disease pathogenesis is unclear. We show that HERV-K is activated in a subpopulation of patients with sporadic amyotrophic lateral sclerosis (ALS) and that its envelope (env) protein may contribute to neurodegeneration. The virus was expressed in cortical and spinal neurons of ALS patients, but not in neurons from control healthy individuals. Expression of HERV-K or its env protein in human neurons caused retraction and beading of neurites. Transgenic animals expressing the env gene developed progressive motor dysfunction accompanied by selective loss of volume of the motor cortex, decreased synaptic activity in pyramidal neurons, dendritic spine abnormalities, nucleolar dysfunction, and DNA damage. Injury to anterior horn cells in the spinal cord was manifested by muscle atrophy and pathological changes consistent with nerve fiber denervation and reinnervation. Expression of HERV-K was regulated by TAR (trans-activation responsive) DNA binding protein 43, which binds to the long terminal repeat region of the virus. Thus, HERV-K expression within neurons of patients with ALS may contribute to neurodegeneration and disease pathogenesis.

The immunosuppressant drug FK506 binds to the immunophilin protein FKBP12 and inhibits its prolyl isomerase activity. Immunosuppressive actions, however, are mediated via an FK506-FKBP12 inhibition of the Ca(2+)-activated phosphatase calcineurin. Physiologic cellular roles for FKBP12 have remained unclear. FKBP12 is physically associated with the RyR and IP3R Ca2+ channels in the absence of FK506, with added FK506 disrupting these complexes. Dissociation of FKBP12 results in alteration of channel Ca2+ conductance in both cases. We now report that calcineurin is physiologically associated with the IP3R-FKBP12 and RyR-FKBP12 receptor complexes and that this interaction can be disrupted by FK506 or rapamycin. Calcineurin anchored to the IP3R via FKBP12 regulates the phosphorylation status of the receptor, resulting in a dynamic Ca(2+)-sensitive regulation of IP3-mediated Ca2+ flux.

Huntington's Disease (HD) is caused by expansion of a CAG repeat within a putative open reading frame of a recently identified gene, IT15. We have examined the expression of the gene's protein product using antibodies developed against the N-terminus and an internal epitope. Both antisera recognize a 350 kDa protein, the predicted size, indicating that the CAG repeat is translated into polyglutamine. The HD protein product is widely expressed, most highly in neurons in the brain. There is no enrichment in the striatum, the site of greatest pathology in HD. Within neurons, the protein is diminished in nuclei and mitochondria and is present in the soluble cytoplasmic compartment, as well as loosely associated with membranes or cytoskeleton, in cell bodies, dendrites, and axons. It is concentrated in nerve terminals, including terminals within the caudate and putamen. Thus, the normal HD gene product may be involved in common intracellular functions, and possibly in regulation of nerve terminal function. The product of the expanded allele is expressed, consistent with a gain of function mechanism for HD at the protein level.

Immunosuppressants FK506 and cyclosporin A inhibit neurotoxicity of N-methyl-D-aspartate in primary cortical cultures, while having no effect on quisqualate-and kainate-mediated neurotoxicity. Rapamycin completely reverses the neuroprotective effect of FK506. Both FK506 and cyclosporin A inhibit NMDA-elicited/nitric oxide-mediated increases in cGMP levels in cortical cultures. FK506 has no effect on sodium nitroprusside-induced increases in cGMP. In a stably transfected human kidney 293 cell line overexpressing the gene encoding nitric oxide synthase [L-arginine, NADPH:oxygen oxidoreductase (nitric oxide-forming), EC 1.14.13.39], FK506 inhibits the calcium ionophore A23187, stimulated increases in nitrite (a breakdown product of nitric oxide), and potentiates phorbol ester-mediated inhibition of nitrite formation. FK506-mediated inhibition of nitric oxide formation is completely reversed by rapamycin. Calcineurin dephosphorylates protein kinase C-mediated phosphorylation of nitric oxide synthase. FK506 prevents the calcineurinmediated dephosphorylation of nitric oxide synthase and thereby diminishes the enzyme's catalytic activity. These data establish nitric oxide synthase as a calcineurin substrate. Nitric oxide synthase catalytic activity is regulated by the phosphorylation state of the enzyme. Enhanced phosphorylation of nitric oxide synthase diminishes catalytic activity, and dephosphorylation (through activation of calcineurin) enhances catalytic activity. The neuroprotective effect of FK506 and cyclosporin A presumably involves the inhibition of calcineurin, preventing the dephosphorylation of nitric oxide synthase and its subsequent activation.Besides their roles in the immune system, the immunophilins cyclophilin and FK506 binding protein (FKBP) are highly concentrated in the brain in discrete neuronal structures where they are colocalized with the Ca2+-activated phosphatase calcineurin (1). Liu et al. (2) demonstrated that very low concentrations of FK506 and cyclosporin A, which bind to FKBP and cyclophilin, respectively, inhibit calcineurin, and we (1) showed that both drugs enhance the phosphorylation of a number of proteins in the brain. Glutamate neurotoxicity acting via N-methyl-D-aspartate (NMDA) receptors is implicated in neuronal damage associated with cerebral infarcts and neurodegenerative diseases (3-5). In primary cerebral cortical cultures (6, 7), in hippocampal slices (8-10), and in animal models of focal ischemia (11) calmodulin-dependent protein kinase, and cGMP-dependent protein kinase (J.L.D., J.P.S., T.M.D., and S.H.S., unpublished data) all inhibit its catalytic activity. Accordingly, we wondered whether enhanced phosphorylation associated with FK506 treatment might alter neurotoxicity. In the present study we demonstrate that low concentrations of FK506 and cyclosporin A markedly diminish NMDA neurotoxicity, enhance the phosphorylation of NOS, inhibit NOS catalytic activity, and inhibit NO-mediated increases in cGMP. MATERIALS AND METHODSCell Cultures. Primary neuronal cul...

We show that the nonimmunosuppressive analogues of the immunosuppressive drugs FK506, rapamycin and cyclosporin A promote neurite outgrowth both in PC12 cells and sensory neuronal cultures of dorsal root ganglia with potencies resembling their immunosuppressive homologues. Neurotrophic potencies of the immunophilin ligands resemble their potencies in binding to and inhibiting the rotamase activity of FKBP-12 of cyclophilin. Since nonimmunosuppressive immunophilin ligands, which are devoid of calcineurin inhibitory activity, are equally neurotrophic, inhibition of calcineurin activity is not the mediator of the neurotrophic effects. The immunophilin ligands are neurotrophic in intact animals. FK506 and L-685,818 (the C18-hydroxy, C21-ethyl derivative of FK506) treatment of rats with crushed sciatic nerves enhances both functional and morphologic recovery. The striking potency of these agents, their bioavailability and the dissociation of neurotrophic from immunosuppressant actions argue for their therapeutic relevance in the treatment of neurodegenerative diseases.

Although immunosuppressant immunophilin ligands promote neurite outgrowth in vitro, their neurotrophic activities are clearly independent of their immunosuppressive activity. In the present report, a novel nonimmunosuppressive immunophilin ligand, GPI-1046 (3-(3-pyridyl)-1-propyl (2S)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrolidinecarboxylate) is described. In vitro, GPI-1046 bound to FK506 binding protein-12 and elicited neurite outgrowth from sensory neuronal cultures with picomolar potency with maximal effects comparable to nerve growth factor. In vivo, GPI-1046 stimulated the regeneration of lesioned sciatic nerve axons and myelin levels. In the central nervous system, GPI-1046 promoted protection and͞or sprouting of serotonincontaining nerve fibers in somatosensory cortex following parachloroamphetamine treatment. GPI-1046 also induced regenerative sprouting from spared nigrostriatal dopaminergic neurons following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity in mice or 6-hydroxydopamine (6-OHDA) toxicity in rats. The rotational abnormality in 6-OHDA treated rats was alleviated by GPI-1046. These neurotrophic actions in multiple models suggest therapeutic utility for GPI-1046 in neurodegenerative diseases.

The immunophilin FK506 binding protein 12 (FKBP12) is associated with and modulates the ryanodine receptor calcium release channel of skeletal muscle. Ryanodine receptor has amino acid homology and functional similarity with another intracellular Ca2+ release channel, the inositol 1,4,5-trisphosphate receptor (IP3R). In the present study we show that highly purified preparations of IP3R contain FKBP12. The complex of these two proteins is disrupted by the immunosuppressants FK506 and rapamycin, both of which are known to bind FKBP12 with high affinity. Disrupting the IP3R-FKBP12 interaction increases Ca2+ flux through IP3R, an effect that is reversed by added FKBP12. FKBP12 appears to be physiologically linked to IP3R, regulating its Ca2+ conductance.

We report the stoichiometric phosphorylation of an inositol 1,4,5-trisphosphate receptor-binding protein from rat brain by the cAMP-dependent protein kinase but not by protein kinase C or Ca2+/calmodulin-dependent protein kinase. This phosphorylation event does not markedly alter [3H]inositol 1,4,5-trisphosphate-binding characteristics. However, inositol 1,4,5-trisphosphate is only 10% as potent in releasing 45Ca2I from phosphorylated, as compared with native, cerebellar microsomes. Phosphorylation of the inositol 1,4,5-trisphosphate-binding protein by the cAMP-dependent protein kinase may provide a biochemical substrate for secondmessenger cross talk.The inositol phospholipid and adenosine 3',5'-cyclic monophosphate (cAMP) systems are major second messengers for effects of neurotransmitters, hormones, and growth factors (1-4). Several lines of evidence imply the existence of cross talk between these two second messengers (5-7). Many