The challenge of follow-on biologics for treatment of multiple sclerosis.

Intellectual property protections for biologic medicinals for multiple sclerosis (MS) are beginning to expire, opening the possibility of development, regulatory approval, and marketing of so-called follow-on biologics, biosimilars, or subsequent entry biologics that might be offered at lower price to consumers and third-party payers, as has been the case for generic drugs. Determining the comparability of a follow-on biologic to its innovator product is more difficult than for small-molecule drugs because of the greater complexity of biologics and the possibility that manufacturing differences can introduce differences in biologic activity and immunogenicity that could result in unpredictable differences in safety or efficacy. We provide a perspective on issues surrounding development, regulatory approval, and potential use of follow-on biologics, with an emphasis on disease-modifying agents for MS.

Neuroimmunophilins: a novel drug therapy for the reversal of neurodegenerative disease?

Neuroimmunophilin ligands (NILs) are drugs derived from the immunosuppressant FK506 (tacrolimus) that have been shown to have variable efficacy in reversing neuronal degeneration and preventing cell death. In a wide range of animal models mimicking Parkinson's disease, dementia and even surgical nerve damage they induce re-sprouting, are neurotrophic or prevent nerve damage. The neurotrophic mechanism of action of these compounds is not known and may be dependent on the type of damage and genetic variability at the species or cellular level. Some evidence suggests that NILs may act through a family of proteins called FK506 binding proteins, some of which may regulate steroid hormone receptors. Other evidence suggests that NILs may protect neurons by upregulating the antioxidant glutathione and stimulating nerve regrowth by inducing the production of neurotrophic factors. Initial clinical trials have had mixed success. In one, patients with moderately severe Parkinson's disease showed no overall improvement in fine motor skills following 6 months of treatment by the neuroimmunophilin GPI 1485. But these patients did exhibit decreased loss of dopaminergic nerve terminals with a low dose of GPI 1485 and in fact some increase in dopaminergic terminals within 6 months of the higher dose of GPI 1485 drug treatment. As a result, a second phase II clinical trial using a patient population with less severe degeneration has been initiated concurrent with an investigation of GPI 1485 and other neuroprotective therapies funded by the National Institute of Neurological Disorders and Stroke. Another clinical trial ongoing at this time is exploring the use of a neuroimmunophilin ligand to prevent nerve degeneration and erectile dysfunction resulting from prostatectomy. In summary, neuroimmunophilins show promise to reverse some forms of neurodegeneration but exact factors that predict outcome have not been identified.

Current status of an antireflux procedure in laparoscopic Heller myotomy.

BACKGROUND: Persistent dysphagia and postoperative gastroesophageal reflux (GER) are the most cited reasons for surgical failure of laparoscopic Heller myotomy. Adding an antireflux procedure to Heller myotomy has been proposed to prevent reflux. We hypothesized that an antireflux procedure added to laparoscopic Heller myotomy has little effect on preventing the symptoms or long-term sequelae of GER in achalasia patients. METHODS: We performed a meta-analysis of studies on human subjects reported in the English language literature from 1991 to 2001 years. RESULTS: An antireflux procedure accompanied laparoscopic myotomy in 15 studies with 532 patients. In 6 studies of 69 patients, no antireflux procedure was added to laparoscopic myotomy. Follow-up was available on 489 patients (92%) with partial fundoplication. The rate of GER diagnosed in pH studies was 7.9% (18 of 228 patients studied), whereas only 5.9% of patients experienced symptoms of GER (29 of 489 patients followed). Of the 69 patients without fundoplication, 47 (68%) were available for follow-up. Forty patients (85%) were studied with pH monitoring postoperatively, with 4 (10%) demonstrating reflux. Six (13%) of 47 patients had symptoms of GER. The difference in the rate of GER diagnosed in postmyotomy pH studies in wrapped and nonwrapped patients was not significant (7.9 vs 10%, respectively; p = 0.75). There was also no significant difference in the incidence of postmyotomy GER symptoms in wrapped and nonwrapped patients (5.9 vs 13% respectively; p = 0.12). CONCLUSIONS: Reflux is not necessarily eliminated with the addition of a partial fundoplication. Based on the published data, recommendations cannot be made regarding the efficacy of adding an antireflux procedure to laparoscopic Heller myotomy. Prospective randomized study is needed to clarify the role of an antireflux procedure after laparoscopic Heller myotomy.

FK506 binding protein 12 is expressed in rat penile innervation and upregulated after cavernous nerve injury.

To evaluate whether FK506 and other immunophilin ligands may have potential therapeutic efficacy for erectile function preservation after penile nerve injury, we demonstrated localizations of the immunophilin FK506 binding protein 12 (FKBP 12) in intact and injured rat penile nerves and correlated these findings with localizations of neuronal nitric oxide synthase (nNOS), which neuronally forms nitric oxide for mediation of penile erection, in response to systemically administered FK506. Adult male Sprague-Dawley rats were subjected to unilateral right cavernous nerve forceps crush injury and administered FK506 (1 mg/kg i.p.) or saline at the same time and daily up to 7 days. At 1, 3 and 7 days after injury, bilateral cavernous nerves and major pelvic ganglia were collected for nNOS immunohistochemistry, FKBP 12 immunohistochemistry, and FKBP 12 in situ hybridisation. Protein expressions of nNOS and FKBP 12 were observed in major pelvic ganglion, cavernous nerve and nerve terminals within the rat penis as well as mRNA expression of FKBP 12 observed in the rat major pelvic ganglion neuronal cell bodies to a minimal extent at baseline conditions. After cavernous nerve injury, nNOS immunoreactivity was observed to be slightly diminished in ipsilateral penile nerve structures at only one day following injury while both FKBP 12 protein and mRNA expressions were observed to be increased at each interval of study. FK506 treatment did not affect staining of intact or injured nerves. Our demonstration that FKBP 12 is localized to penile innervation in the rat and becomes upregulated following cavernous nerve crush injury, independent of FK506 treatment, suggests that this immunophilin mediates a neurotrophic mechanism. Whether FK506 affords neuroprotection that preserves penile erection through FKBP 12 upregulation is unclear.

The non-immunosuppressive immunophilin ligand GPI-1046 potently stimulates regenerating axon growth from adult mouse dorsal root ganglia cultured in Matrigel.

We used explant cultures of adult mouse dorsal root ganglia with spinal nerve attached growing in Matrigel to assess the effects of the non-immunosuppressive immunophilin ligand GPI-1046 [Snyder et al. (1998) TIPS 19, 21-26] on the growth rate of regenerating sensory axons and found a potent stimulation of axon growth. In these explant cultures, naked, unfasciculated axons emerge from the cut end of the spinal nerve and continue to grow in the Matrigel for up to eight days [Tonge et al. (1996) Neuroscience 73, 541-551]. Some axons are entirely smooth whilst others show prominent varicosities. Some of the former express the phosphorylated neurofilament epitope recognised by monoclonal antibody RT97, a marker for large calibre, myelinated axons, whilst the latter express calcitonin gene-related peptide, predominantly a marker for unmyelinated, and small diameter myelinated sensory axons. Many of the axons in these cultures also express the low-affinity neurotrophin receptor p75. GPI-1046 has been shown to have striking stimulatory effects on embryonic primary sensory axons growing in vitro and it was therefore of interest to see whether it could also enhance regenerating sensory axon growth from the adult ganglia in our cultures. GPI-1046 potently stimulated axon growth in our cultures in a dose-dependent manner. The stimulatory effect was not dependent on the class of sensory axon. These observations show that GPI-1046 is a potent stimulator of regenerating axons from adult, primary sensory neurones. The cellular site of action of GPI-1046 is unknown. To distinguish between a direct effect of the drug on neurones and an indirect effect we compared the effects of GPI-1046 on explant and dissociated cultures. In confirmation of previous results, we found that GPI-1046 potently stimulated axon outgrowth from explants of embryonic chick dorsal root ganglia. However, the drug was without effect on dissociated embryonic dorsal root ganglion neurones, suggesting that non-neuronal cells are important for axon growth stimulation.

Regeneration of dopaminergic function in 6-hydroxydopamine-lesioned rats by neuroimmunophilin ligand treatment.

Nonimmunosuppressant immunophilin ligands have been found previously to stimulate neurite growth in culture and to promote regeneration of peripheral and central nerve fibers in vivo. To further characterize the effectiveness of these ligands, we have investigated the effect of the immunophilin ligand GPI-1046 in 6-hydroxydopamine (6-OHDA)-lesioned rats. In unlesioned rats, tetanic stimulation of the white matter induced long-term potentiation (LTP) of corticostriatal synaptic transmission as indicated by a 40-100% increase in the field potential amplitudes recorded in striatal brain slices. Unilateral microinjection of 6-OHDA into the substantia nigra resulted in a loss of corticostriatal LTP and in significant abnormality of motor behavior as assessed by amphetamine-induced ipsilateral rotations. Daily treatment of 6-OHDA-lesioned rats with GPI-1046 (10 mg/kg, s.c.) for 1 week reduced amphetamine-induced rotations by 75% and greatly restored the striatal tyrosine hydroxylase immunostaining. In addition, GPI-1046 almost completely restored corticostriatal LTP in 6-OHDA-lesioned animals. LTP in normal animals and that restored by GPI-1046 in lesioned animals were both blocked by the NMDA receptor antagonist APV, suggesting mediation by NMDA receptors. Both LTPs were sensitive to dopamine (DA) receptor antagonists. The nonselective DA receptor antagonist chlorpromazine and the selective D1-D5 receptor antagonist SCH23390 reduced the LTP by 90%. These results demonstrate that the immunophilin ligand GPI-1046 can reverse the abnormalities in the substantia nigra-striatal dopaminergic system that are caused by 6-OHDA, thus providing a potential therapeutic agent for Parkinson's disease.

Systemic administration of the immunophilin ligand GPI 1046 in MPTP-treated monkeys.

Systemic administration of immunophilin ligands provides trophic influences to dopaminergic neurons in rodent models of Parkinson's disease (PD) resulting in the initiation of clinical trials in patients with Parkinson's disease. We believe that prior to clinical trials, novel therapeutic strategies should show safety and efficacy in nonhuman models of PD. The present study assessed whether oral administration of the immunophilin 3-(3-pyridyl)-1-propyl (2S)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrollidinecarboxylate (GPI 1046) could prevent the structural and functional consequences of n-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration in nonhuman primates. Twenty-five rhesus monkeys received daily oral administration of vehicle (n = 5) or one of four doses of GPI 1046 (0.3 mg/kg, n = 5; 1.0 mg/kg, n = 5; 3.0 mg/kg, n = 5; 10.0 mg/kg, n = 5). Two weeks after starting the drug treatment, all monkeys received a unilateral intracarotid injection of MPTP-HCl (3 mg). Daily drug administration continue for 6 weeks postlesion after which time the monkeys were sacrificed. Monkeys were assessed for performance on a hand reach task, general activity, and clinical dysfunction based on a clinical rating scale. All groups of monkeys displayed similar deficits on each behavioral measure as well as similar losses of tyrosine hydroxylase (TH)-immunoreactive (ir) nigral neurons, TH-mRNA, and TH-ir striatal optical density indicating that in general treatment failed to have neuroprotective effects.

The small molecule FKBP ligand GPI 1046 induces partial striatal re-innervation after intranigral 6-hydroxydopamine lesion in rats.

Extensive unilateral striatal deafferentation was produced by intranigral 6-hydroxydopamine (6-OHDA) in rats. Beginning 60 days after 6-OHDA injection animals received a 14-day course of treatment with either the small molecule FKBP ligand GPI 1046 (10 mg/kg) or its vehicle alone. Striatal dopaminergic innervation density was determined from high power image analysis of striatal tyrosine hydroxylase (TH) immunohistochemistry. GPI 1046 treatment did not alter TH fiber density in the contralateral striatum but did produce significantly higher striatal TH fiber density in the ipsilateral caudate-putamen. This striatal re-innervation occurred in the absence of increased nigral sparing, and appears to reflect the GPI 1046 induced sprouting of residual TH+ fibers spared by the 6-OHDA lesion.

Systemic treatment with GPI 1046 improves spatial memory and reverses cholinergic neuron atrophy in the medial septal nucleus of aged mice.

Systemic treatment with GPI 1046, a non-immunosuppressive ligand of the immunophilin FKBP12 (FK-506-binding protein 12 kDa), has previously been shown to promote morphological recovery of the nigrostriatal dopaminergic projection after MPTP lesion in mice, and of lesioned sciatic nerve fibres after nerve crush in rats. In the present study, we investigated whether chronic systemic treatment with GPI 1046 could affect the decline of spatial learning and memory, and the atrophy of medial septal cholinergic neurons, associated with late senescence in C57 black mice. Three-month old (young) and 18-19-month old (aged) male C57BL/6N-Nia mice were first trained in a place learning task in the Morris water maze. Based on their performance relative to young controls, aged animals were then allocated to treatment groups (10 mg/kg GPI 1046, or vehicle). Retention of the spatial platform location was assessed after 3 weeks of dosing. We found that aged animals that had been dosed with GPI 1046 now performed at a significantly better level than their vehicle control group. Aged animals that had shown the greatest degree of impairment during training in the place learning task showed the greatest relative degree of improvement under treatment and were statistically indistinguishable from young, or aged unimpaired control animals. Cell volumes of cholinergic cells in the medial septal nucleus were assessed after an additional 10 months of dosing at 30 months of age, using stereological methods. We found that aged animals displayed a significant 34% decrease in volume of these cells relative to young controls. This atrophy was significantly reversed in aged GPI 1046-treated animals (13% shrinkage). We conclude that chronic systemic treatment with GPI 1046 positively affects memory mechanisms in the aged mouse, possibly by acting on the septohippocampal cholinergic system.

Dopamine transporter loss and clinical changes in MPTP-lesioned primates.

Single photon emission computed tomography (SPECT) and the dopamine (DA) transporter tracer, 2 beta-carboxymethoxy-3 beta-(4-iodophenyl)tropane ([123I]beta-CIT), were used to determine DA transporter density in 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP)-lesioned monkeys with varying degrees of parkinsonism. The clinical stage of parkinsonism corresponded to SPECT measures of striatal DA transporter density suggesting that more severe parkinsonism was associated with a greater degree of dopaminergic terminal degeneration. These findings are similar to those reported earlier using positron emission tomography (PET) and the DA metabolism tracer, 6-[18F]fluoro-L-m-tyrosine (FMT), indicating that both are good methods for evaluating nigrostriatal degeneration in MPTP primate models.

The 12 kD FK 506 binding protein FKBP12 is released in the male reproductive tract and stimulates sperm motility.

BACKGROUND: The 12 kD FK506 binding protein FKBP12 is a cytosolic receptor for the immunosuppressant drugs FK506 and rapamycin. In addition to its critical role in drug-induced T-cell immunosuppression, FKBP12 associates physiologically with ryanodine and inositol 1,4,5-trisphosphate (IP3) receptors, regulating their ability to flux calcium. We investigated a role for FKBP12 in male reproductive physiology on the basis of our identification of extremely high levels of [3H]FK506 binding in male reproductive tissues. MATERIALS AND METHODS: [3H]FK506 binding studies were performed to identify tissues enriched with FK506 binding sites. The abundant [3H]FK506 binding sites identified in the male reproductive tract were localized by [3H]FK506 autoradiography. FK506 affinity chromatography was employed to purify FKBP from epididymal fluid. Anti-FKBP12 Western analysis was used to confirm the identity of the purified FKBP. The binding of exogenous FKBP12 to sperm was evaluated by [32P]FKBP12 binding studies and [33P]FKBP12 autoradiography. The effect of recombinant FKBP12 on sperm motility was investigated using a Hamilton Thorne motility analyzer. RESULTS: Male reproductive tissues contained high levels of [3H]FK506 binding. The localization of [3H]FK506 binding sites to the tubular epithelium of the caput epididymis and the lumen of the cauda and vas deferens suggested that FKBP is released in the male reproductive tract. FKBP12 was purified from epididymal plasma by FK506 affinity chromatography. Radiolabeled FKBP12 specifically bound to immature but not mature sperm. In sperm motility studies, FKBP12-treated caput sperm exhibited double the curvilinear velocity of untreated controls. CONCLUSIONS: High levels of FKBP12 are released in the male reproductive tract and specifically associate with maturing sperm. Recombinant FKBP12 enhances the curvilinear velocity of immature sperm, suggesting a role for FKBP12 in motility initiation. The highest concentrations of soluble FKBP12 in the male reproductive tract occur in the lumen of the vas deferens, a site of sperm storage and the conduit for ejaculated sperm. Preservation of mammalian sperm for reproductive technologies may be optimized by supplementing incubation or storage media with FKBP12.

Neural actions of immunophilin ligands.

Immunophilins, protein receptors for immunosuppressant drugs such as cyclosporin A and FK506, are enriched far more in the brain than in the immune system. Drug-immunophilin complexes bind to calcineurin, inhibiting its phosphatase activity and leading to immunosuppressant effects. The immunophilin FKBP-12 (FK506 binding protein, 12 kDa) forms a complex with the ryanodine and inositol (1,4,5) trisphosphate (IP3) receptors to regulate their physiological release of intracellular Ca2+. Here, Solomon Snyder and colleagues describe how non-immunosuppressant as well as immunosuppressant immunophilin ligands are neurotrophic for numerous classes of damaged neurones, both in culture systems and intact animals. Their ability to stimulate functional regrowth of damaged sciatic, cortical cholinergic, dopamine and 5-HT neurones may have therapeutic relevance.

Neurotrophic actions of nonimmunosuppressive analogues of immunosuppressive drugs FK506, rapamycin and cyclosporin A.

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.

Neurotrophic immunophilin ligands stimulate structural and functional recovery in neurodegenerative animal models.

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 and/or sprouting of serotonin-containing 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.

Immunophilin regulation of neurotransmitter release.

BACKGROUND: The immunophilins are proteins that mediate actions of immunosuppressant drugs such as FK506 and cyclosporin A by binding to calcineurin, inhibiting its phosphatase activity, and increasing the phosphorylation level of transcription factors required for interleukin 2 formation. Though concentrations in the brain greatly exceed levels in immune tissues, no function has been previously established for nervous system immunophilins. Nitric oxide (NO) has been implicated in neurotransmitter release. FK506 appears to inhibit NO production by maintaining NO synthase in a highly phosphorylated and thereby inactivated state. Accordingly, we examined effects of FK506 and cyclosporin A on neurotransmitter release in PC12 cells treated with nerve growth factor (NGF) and in rat brain striatal synaptosomes. MATERIALS AND METHODS: We monitored effects of immunophilin ligands on [3H]-neurotransmitter release from PC12 cells differentiated with NGF. Rat brain striatal synaptosomes were loaded with radiolabeled transmitters and treated with FK506 or cyclosporin A prior to initiating neurotransmitter release with N-methyl-D-aspartate (NMDA) or potassium depolarization. Striatal synaptosomes were also loaded with 32P-orthophosphate and treated with FK506. 32P-labeled synaptic vesicle proteins were isolated from these synaptosomes in an attempt to relate specific FK506-dependent phosphorylation of vesicle proteins with the effects of FK506 on neurotransmitter release. Identification of proteins targetted by FK506 was made by immunoblot analysis and immunoprecipitation. RESULTS: Low nanomolar concentrations of the immunosuppressant drugs FK506 and cyclosporin A (CsA) inhibit transmitter release from PC-12 cells and from NMDA-stimulated brain synaptosomes. By contrast, the immunosuppressants augment depolarization-induced transmitter release from synaptosomes. Synapsin I, a synaptic vesicle phosphoprotein, displays enhanced phosphorylation in the presence of FK506. CONCLUSIONS: Inhibition of transmitter release in PC-12 cells and NMDA-treated synaptosomes by immunosuppressants may reflect augmented phosphorylation of NO synthase, reducing its catalytic activity. This fits with the requirement of NO for transmitter release in PC12 cells and NMDA-treated synaptosomes. Stimulation by immunosuppressants of transmitter release in potassium depolarized synaptosomes may result from augmented phosphorylation of synapsin I, whose phosphorylation is known to facilitate transmitter release. Thus, immunophilins may modulate release of numerous neurotransmitters both by influencing NO formation and the phosphorylation state of synaptic vesicle-associated proteins.

Calcineurin associated with the inositol 1,4,5-trisphosphate receptor-FKBP12 complex modulates Ca2+ flux.

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.

Nitric oxide synthase, immunophilins and poly(ADP-ribose) synthetase: novel targets for the development of neuroprotective drugs.

During ischemic stroke, massive neural damage occurs due to excess release of glutamate which acts mainly through N-methyl-D-aspartate (NMDA) receptors. Activation of the NMDA receptor stimulates nitric oxide (NO) production by NO synthase (NOS). NO mediates glutamate neurotoxicity as inhibitors of NOS prevent neuronal death. FK506, an immunosuppressant drug, binds to FK506 binding protein (FKBP). One target of the FK506/FKBP complex is the calcium/calmodulin-dependent protein phosphatase calcineurin, whose activity is inhibited upon interaction with FK506/FKBP. FK506 treatment increases phosphorylation level of calcinurin substrates including NOS. As a potent neuroprotective agent in vitro and in vivo, FK506 increases NOS phosphorylation and decreases NO production. NO activates poly(ADP-ribose) synthetase (PARS), a nuclear enzyme that synthesizes poly(ADP-ribose) from NAD. Prolonged activation of PARS depletes NAD and lowers cellular energy levels. Inhibition of PARS also prevents NO toxicity. NOS inhibitors, immunosuppressants and PARS inhibitors may be useful agents to prevent neuronal damage during stroke.

Widespread expression of Huntington's disease gene (IT15) protein product.

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.

Neuronal regeneration enhances the expression of the immunophilin FKBP-12.

Immunophilins are a group of proteins that serve as receptors for the immunosuppressant drugs cyclosporin A and FK506. The immunophilin designated FK-506 binding protein-12 (FKBP-12) is concentrated more than 10 times higher in the brain than in immune tissues. The complex of FK506 and FKBP-12 inhibits the calcium activated phosphatase, calcineurin, increasing phosphorylated levels of calcineurin substrates with growth associated protein-43 (GAP-43), being most prominent in the brain. We now demonstrate an association of FKBP-12 with neuronal regeneration and GAP-43 disposition. Facial nerve crush markedly augments expression of FKBP-12 mRNA in the facial nucleus with a time course paralleling changes in GAP-43 mRNA. Following sciatic nerve lesions, similar increases in FKBP-12 mRNA occur in lumbar motor neurons and dorsal root ganglia neuronal cells. Increased FKBP-12 expression appears linked to regeneration rather than degeneration as facial nerve lesions elicited by ricin injection, which produce neuronal death without regeneration, fail to augment FKBP-12 expression in the facial nucleus. The time course for accumulation of FKBP-12 in sciatic nerve segments following nerve crush indicates rapid axonal transport at a rate similar to GAP-43.