Search

ATTR amyloidosis is a systemic, debilitating and fatal disease caused by transthyretin (TTR) amyloid accumulation. RNA interference (RNAi) is a clinically validated technology that may be a promising approach to the treatment of ATTR amyloidosis. The vast majority of TTR, the soluble precursor of TTR amyloid, is expressed and synthesized in the liver. RNAi technology enables robust hepatic gene silencing, the goal of which would be to reduce systemic levels of TTR and mitigate many of the clinical manifestations of ATTR that arise from hepatic TTR expression. To test this hypothesis, TTR-targeting siRNAs were evaluated in a murine model of hereditary ATTR amyloidosis. RNAi-mediated silencing of hepatic TTR expression inhibited TTR deposition and facilitated regression of existing TTR deposits in pathologically relevant tissues. Further, the extent of deposit regression correlated with the level of RNAi-mediated knockdown. In comparison to the TTR stabilizer, tafamidis, RNAi-mediated TTR knockdown led to greater regression of TTR deposits across a broader range of affected tissues. Together, the data presented herein support the therapeutic hypothesis behind TTR lowering and highlight the potential of RNAi in the treatment of patients afflicted with ATTR amyloidosis.

One possible treatment for Huntington's disease involves direct infusion of a small, interfering RNA (siRNA) designed to reduce huntingtin expression into brain tissue from a chronically implanted programmable pump. Here, we studied the suppression of huntingtin mRNA achievable with short infusion times, and investigated how long suppression may persist after infusion ceases. Rhesus monkeys received 3 days of infusion of Magnevist into the putamen to confirm catheter patency and fluid distribution. After a 1-week washout period, monkeys received radiolabeled siRNA targeting huntingtin. After 1 or 3 days of siRNA delivery, monkeys were either terminated, or their pumps were shut off and they were terminated 10 or 24 days later. Results indicate that the onset of huntingtin mRNA suppression in the rhesus putamen occurs rapidly, achieving a plateau throughout the putamen within 4 days. Conversely, loss of huntingtin suppression progresses slowly, persisting an estimated 27-39 days in the putamen and surrounding white matter. These findings indicate the rapid onset and durability of siRNA-mediated target gene suppression observed in other organs also occurs in the brain, and support the use of episodic delivery of siRNA into the brain for treatment of Huntington's disease and possibly other neurodegenerative diseases.

Pharmacologic target gene modulation is the primary objective for RNA antagonist strategies and gene therapy. Here we show that mRNAs encoding tissue-specific gene transcripts can be detected in biological fluids and that RNAi-mediated target gene silencing in the liver and brain results in quantitative reductions in serum and cerebrospinal fluid mRNA levels, respectively. Further, administration of an anti-miRNA oligonucleotide resulted in decreased levels of the miRNA in circulation. Moreover, ectopic expression of an adenoviral transgene in the liver was quantified based on measurement of serum mRNA levels. This noninvasive method for monitoring tissue-specific RNA modulation could greatly advance the clinical development of RNA-based therapeutics.

Background: Transthyretin amyloidosis is caused by the deposition of hepatocyte-derived transthyretin amyloid in peripheral nerves and the heart. A therapeutic approach mediated by RNA interference (RNAi) could reduce the production of transthyretin., Methods: We identified a potent antitransthyretin small interfering RNA, which was encapsulated in two distinct first- and second-generation formulations of lipid nanoparticles, generating ALN-TTR01 and ALN-TTR02, respectively. Each formulation was studied in a single-dose, placebo-controlled phase 1 trial to assess safety and effect on transthyretin levels. We first evaluated ALN-TTR01 (at doses of 0.01 to 1.0 mg per kilogram of body weight) in 32 patients with transthyretin amyloidosis and then evaluated ALN-TTR02 (at doses of 0.01 to 0.5 mg per kilogram) in 17 healthy volunteers., Results: Rapid, dose-dependent, and durable lowering of transthyretin levels was observed in the two trials. At a dose of 1.0 mg per kilogram, ALN-TTR01 suppressed transthyretin, with a mean reduction at day 7 of 38%, as compared with placebo (P=0.01); levels of mutant and nonmutant forms of transthyretin were lowered to a similar extent. For ALN-TTR02, the mean reductions in transthyretin levels at doses of 0.15 to 0.3 mg per kilogram ranged from 82.3 to 86.8%, with reductions of 56.6 to 67.1% at 28 days (P<0.001 for all comparisons). These reductions were shown to be RNAi-mediated. Mild-to-moderate infusion-related reactions occurred in 20.8% and 7.7% of participants receiving ALN-TTR01 and ALN-TTR02, respectively., Conclusions: ALN-TTR01 and ALN-TTR02 suppressed the production of both mutant and nonmutant forms of transthyretin, establishing proof of concept for RNAi therapy targeting messenger RNA transcribed from a disease-causing gene. (Funded by Alnylam Pharmaceuticals; ClinicalTrials.gov numbers, NCT01148953 and NCT01559077.).

Unlabelled: RNA interference (RNAi) is a potent and specific mechanism for regulating gene expression. Harnessing RNAi to silence genes involved in disease holds promise for the development of a new class of therapeutics. Delivery is key to realizing the potential of RNAi, and lipid nanoparticles (LNP) have proved effective in delivery of siRNAs to the liver and to tumors in animals. To examine the activity and safety of LNP-formulated siRNAs in humans, we initiated a trial of ALN-VSP, an LNP formulation of siRNAs targeting VEGF and kinesin spindle protein (KSP), in patients with cancer. Here, we show detection of drug in tumor biopsies, siRNA-mediated mRNA cleavage in the liver, pharmacodynamics suggestive of target downregulation, and antitumor activity, including complete regression of liver metastases in endometrial cancer. In addition, we show that biweekly intravenous administration of ALN-VSP was safe and well tolerated. These data provide proof-of-concept for RNAi therapeutics in humans and form the basis for further development in cancer., Significance: The fi ndings in this report show safety, pharmacokinetics, RNAi mechanism of action, and clinical activity with a novel fi rst-in-class LNP-formulated RNAi therapeutic in patients with cancer. The ability to harness RNAi to facilitate specifi c multitargeting, as well as increase the number of druggable targets, has important implications for future drug development in oncology., (©2012 AACR.)

RNA interference is a promising strategy for the treatment of Huntington's disease (HD) as it can specifically decrease the expression of the mutant Huntingtin protein (Htt). However, siRNA does not cross the blood-brain barrier and therefore delivery to the brain is limited to direct CNS delivery. Non-invasive delivery of siRNA through the blood-brain barrier (BBB) would be a significant advantage for translating this therapy to HD patients. Focused ultrasound (FUS), combined with intravascular delivery of microbubble contrast agent, was used to locally and transiently disrupt the BBB in the right striatum of adult rats. 48h following treatment with siRNA, the right (treated) and the left (control) striatum were dissected and analyzed for Htt mRNA levels. We demonstrate that FUS can non-invasively deliver siRNA-Htt directly to the striatum leading to a significant reduction of Htt expression in a dose dependent manner. Furthermore, we show that reduction of Htt with siRNA-Htt was greater when the extent of BBB disruption was increased. This study demonstrates that siRNA treatment for knockdown of mutant Htt is feasible without the surgical intervention previously required for direct delivery to the brain., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Sustaining a high growth rate requires tumors to exploit resources in their microenvironment. One example of this is the extensive angiogenesis that is a typical feature of high-grade gliomas. Here, we show that expression of the constitutively active mutant epidermal growth factor receptor, ΔEGFR (EGFRvIII, EGFR*, de2-7EGFR) is associated with significantly higher expression levels of the pro-angiogenic factor interleukin (IL)-8 in human glioma specimens and glioma stem cells. Furthermore, the ectopic expression of ΔEGFR in different glioma cell lines caused up to 60-fold increases in the secretion of IL-8. Xenografts of these cells exhibit increased neovascularization, which is not elicited by cells overexpressing wild-type (wt)EGFR or ΔEGFR with an additional kinase domain mutation. Analysis of the regulation of IL-8 by site-directed mutagenesis of its promoter showed that ΔEGFR regulates its expression through the transcription factors nuclear factor (NF)-κB, activator protein 1 (AP-1) and CCAAT/enhancer binding protein (C/EBP). Glioma cells overexpressing ΔEGFR showed constitutive activation and DNA binding of NF-κB, overexpression of c-Jun and activation of its upstream kinase c-Jun N-terminal kinase (JNK) and overexpression of C/EBPβ. Selective pharmacological or genetic targeting of the NF-κB or AP-1 pathways efficiently blocked promoter activity and secretion of IL-8. Moreover, RNA interference-mediated knock-down of either IL-8 or the NF-κB subunit p65, in ΔEGFR-expressing cells attenuated their ability to form tumors and to induce angiogenesis when injected subcutaneously into nude mice. On the contrary, the overexpression of IL-8 in glioma cells lacking ΔEGFR potently enhanced their tumorigenicity and produced highly vascularized tumors, suggesting the importance of this cytokine and its transcription regulators in promoting glioma angiogenesis and tumor growth.

Therapeutics based on RNA interference (RNAi) have emerged as a potential new class of drugs for treating human disease by silencing the target messenger RNA (mRNA), thereby reducing levels of the corresponding pathogenic protein. The major challenge for RNAi therapeutics is the development of safe delivery vehicles for small interfering RNAs (siRNAs). We previously showed that cholesterol-conjugated siRNAs (chol-siRNA) associate with plasma lipoprotein particles and distribute primarily to the liver after systemic administration to mice. We further demonstrated enhancement of silencing by administration of chol-siRNA pre-associated with isolated high-density lipoprotein (HDL) or low-density lipoprotein (LDL). In this study, we investigated mimetic lipoprotein particle prepared from recombinant apolipoprotein A1 (apoA) and apolipoprotein E3 (apoE) as a delivery vehicle for chol-siRNAs. We show that apoE-containing particle (E-lip) is highly effective in functional delivery of chol-siRNA to mouse liver. E-lip delivery was found to be considerably more potent than apoA-containing particle (A-lip). Furthermore, E-lip-mediated delivery was not significantly affected by high endogenous levels of plasma LDL. These results demonstrate that E-lip has substantial potential as delivery vehicles for lipophilic conjugates of siRNAs.

RNA interference (RNAi) is a sequence-specific gene-silencing mechanism triggered by double-stranded RNA and powerful tools for a gene function study and RNAi therapy. Although siRNAs offer several advantages as potential new drugs to treat various diseases, the efficient delivery system of siRNAs in vivo remains a crucial challenge for achieving the desired RNAi effect in clinical development. In particular, when considering the siRNA therapeutics for familial amyloidotic polyneuropathy (FAP) caused by the deposition of variant transthyretin (TTR) in various organs, hepatocyte-selective siRNA delivery is desired because TTR is predominantly synthesized by hepatocytes. In this study, to reveal the potential use of lactosylated dendrimer (G3)/α-cyclodextrin conjugate (Lac-α-CDE (G3)) as novel hepatocyte-selective siRNA carriers in order to treat FAP, we evaluated the RNAi effect of siRNA complex with Lac-α-CDE (G3) both in vitro and in vivo.

Huntington's disease is an autosomal dominant neurodegenerative disease caused by a toxic gain of function mutation in the huntingtin gene (Htt). Silencing of Htt with RNA interference using direct CNS delivery in rodent models of Huntington's disease has been shown to reduce pathology and promote neuronal recovery. A key translational step for this approach is extension to the larger non-human primate brain, achieving sufficient distribution of small interfering RNA targeting Htt (siHtt) and levels of Htt suppression that may have therapeutic benefit. We evaluated the potential for convection enhanced delivery (CED) of siHtt to provide widespread and robust suppression of Htt in nonhuman primates. siHtt was infused continuously for 7 or 28 days into the nonhuman primate putamen to analyze effects of infusion rate and drug concentration on the volume of effective suppression. Distribution of radiolabeled siHtt and Htt suppression were quantified by autoradiography and PCR, respectively, in tissue punches. Histopathology was evaluated and Htt suppression was also visualized in animals treated for 28 days. Seven days of CED led to widespread distribution of siHtt and significant Htt silencing throughout the nonhuman primate striatum in an infusion rate and dose dependent manner. Htt suppression at therapeutic dose levels was well tolerated by the brain. A model developed from these results predicts that continuous CED of siHtt can achieve significant coverage of the striatum of Huntington's disease patients. These findings suggest that this approach may provide an important therapeutic strategy for treating Huntington's disease., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Huntington disease is an autosomal dominant neurodegenerative disorder caused by a toxic expansion in the CAG repeat region of the huntingtin gene. Oligonucleotide approaches based on RNAi and antisense oligonucleotides provide promising new therapeutic strategies for direct intervention through reduced production of the causative mutant protein. Allele-specific and simultaneous mutant and wild-type allele-lowering strategies are being pursued with local delivery to the brain, each with relative merits. Delivery remains a key challenge for translational success, especially with chronic therapy. The potential of disease-modifying oligonucleotide approaches for Huntington disease will be revealed as they progress into clinical trials.

Human solid tumors frequently have pronounced heterogeneity of both neoplastic and normal cells on the histological, genetic, and gene expression levels. While current efforts are focused on understanding heterotypic interactions between tumor cells and surrounding normal cells, much less is known about the interactions between and among heterogeneous tumor cells within a neoplasm. In glioblastoma multiforme (GBM), epidermal growth factor receptor gene (EGFR) amplification and mutation (EGFRvIII/DeltaEGFR) are signature pathogenetic events that are invariably expressed in a heterogeneous manner. Strikingly, despite its greater biological activity than wild-type EGFR (wtEGFR), individual GBM tumors expressing both amplified receptors typically express wtEGFR in far greater abundance than the DeltaEGFR lesion. We hypothesized that the minor DeltaEGFR-expressing subpopulation enhances tumorigenicity of the entire tumor cell population, and thereby maintains heterogeneity of expression of the two receptor forms in different cells. Using mixtures of glioma cells as well as immortalized murine astrocytes, we demonstrate that a paracrine mechanism driven by DeltaEGFR is the primary means for recruiting wtEGFR-expressing cells into accelerated proliferation in vivo. We determined that human glioma tissues, glioma cell lines, glioma stem cells, and immortalized mouse Ink4a/Arf(-/-) astrocytes that express DeltaEGFR each also express IL-6 and/or leukemia inhibitory factor (LIF) cytokines. These cytokines activate gp130, which in turn activates wtEGFR in neighboring cells, leading to enhanced rates of tumor growth. Ablating IL-6, LIF, or gp130 uncouples this cellular cross-talk, and potently attenuates tumor growth enhancement. These findings support the view that a minor tumor cell population can potently drive accelerated growth of the entire tumor mass, and thereby actively maintain tumor cell heterogeneity within a tumor mass. Such interactions between genetically dissimilar cancer cells could provide novel points of therapeutic intervention.

Lipid nanoparticles (LNPs) have proven to be highly efficient carriers of short-interfering RNAs (siRNAs) to hepatocytes in vivo; however, the precise mechanism by which this efficient delivery occurs has yet to be elucidated. We found that apolipoprotein E (apoE), which plays a major role in the clearance and hepatocellular uptake of physiological lipoproteins, also acts as an endogenous targeting ligand for ionizable LNPs (iLNPs), but not cationic LNPs (cLNPs). The role of apoE was investigated using both in vitro studies employing recombinant apoE and in vivo studies in wild-type and apoE(-/-) mice. Receptor dependence was explored in vitro and in vivo using low-density lipoprotein receptor (LDLR(-/-))-deficient mice. As an alternative to endogenous apoE-based targeting, we developed a targeting approach using an exogenous ligand containing a multivalent N-acetylgalactosamine (GalNAc)-cluster, which binds with high affinity to the asialoglycoprotein receptor (ASGPR) expressed on hepatocytes. Both apoE-based endogenous and GalNAc-based exogenous targeting appear to be highly effective strategies for the delivery of iLNPs to liver.

Conjugation of small interfering RNA (siRNA) with lipophilic molecules has been demonstrated to enhance cellular uptake in cell culture and to produce efficient endogenous gene silencing in the liver after systemic administration and in neurons after direct local injection. Here, we evaluated the in vivo delivery of siRNAs conjugated with different linkers to cholesterol by targeting CNPase (2'-3'-cyclic nucleotide 3'-phosphodiesterase) in oligodendrocytes. Cholesterol-conjugated siRNAs administered to the rat corpus callosum by intraparenchymal central nervous system (CNS) infusion show improved silencing ability compared with unconjugated siRNA. Furthermore, conjugation of siRNA to cholesterol with a cleavable disulfide linker appears to be beneficial for improving the potency of silencing of CNPase mRNA in oligodendrocytes in vivo. Taken together, these findings indicate that cholesterol-conjugated siRNAs are effective for direct CNS delivery to oligodendrocytes, and that the biocleavable disulfide linker appears to be beneficial for improving the potency of silencing of target mRNA in vivo., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Significant effort has been applied to discover and develop vehicles which can guide small interfering RNAs (siRNA) through the many barriers guarding the interior of target cells. While studies have demonstrated the potential of gene silencing in vivo, improvements in delivery efficacy are required to fulfill the broadest potential of RNA interference therapeutics. Through the combinatorial synthesis and screening of a different class of materials, a formulation has been identified that enables siRNA-directed liver gene silencing in mice at doses below 0.01 mg/kg. This formulation was also shown to specifically inhibit expression of five hepatic genes simultaneously, after a single injection. The potential of this formulation was further validated in nonhuman primates, where high levels of knockdown of the clinically relevant gene transthyretin was observed at doses as low as 0.03 mg/kg. To our knowledge, this formulation facilitates gene silencing at orders-of-magnitude lower doses than required by any previously described siRNA liver delivery system.

We adopted a rational approach to design cationic lipids for use in formulations to deliver small interfering RNA (siRNA). Starting with the ionizable cationic lipid 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), a key lipid component of stable nucleic acid lipid particles (SNALP) as a benchmark, we used the proposed in vivo mechanism of action of ionizable cationic lipids to guide the design of DLinDMA-based lipids with superior delivery capacity. The best-performing lipid recovered after screening (DLin-KC2-DMA) was formulated and characterized in SNALP and demonstrated to have in vivo activity at siRNA doses as low as 0.01 mg/kg in rodents and 0.1 mg/kg in nonhuman primates. To our knowledge, this represents a substantial improvement over previous reports of in vivo endogenous hepatic gene silencing.

The gene for phosphatidylinositol-4-phosphate adaptor-2 (FAPP2) encodes a cytoplasmic lipid transferase with a plekstrin homology domain that has been implicated in vesicle maturation and transport from trans-Golgi to the plasma membrane. The introduction of ribozymes targeting the FAPP2 gene in colon carcinoma cells induced their apoptosis in the presence of Fas agonistic antibody. Furthermore, by quantitative PCR we showed that a siRNA specific to FAPP2, but not a randomized siRNA control, reduced FAPP2 gene expression in tumor cells. Transfection of FAPP2 siRNA into human tumor cells then incubated with FasL resulted in reduction of viable cell numbers. Also, FAPP2 siRNA transfected glioma and breast tumor cells showed significant increases in apoptosis upon incubation with soluble FasL, but the apoptosis did not necessarily correlate with increased Fas expression. These data demonstrate a previously unknown role for FAPP2 in conferring resistance to apoptosis and indicate that FAPP2 may be a target for cancer therapy.

The most significant challenge remaining in the development of small interfering RNAs (siRNAs) as a new class of therapeutic drugs is successful delivery in vivo. The majority of reported studies describing delivery of siRNA or short hairpin RNA (shRNA) to the central nervous system (CNS) have focused on RNA interference (RNAi) in neurons. Here we show direct CNS delivery of siRNA to a different cell type-oligodendrocytes-using convection-enhanced delivery, and demonstrate robust silencing of an endogenous oligodendrocyte-specific gene, 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) with siRNA formulated in saline. The silencing is not sequence-dependent as several different siRNAs are effective in inhibiting target gene expression. Furthermore, we show that CNPase mRNA reduction is dose-dependent, durable for up to 1 week, and mediated by an RNAi mechanism. Increasing the flow rate of siRNA infusion increased the distribution of mRNA suppression to encompass white matter regions distant from the infusion site. Finally, we demonstrate suppression of CNPase mRNA in the nonhuman primate CNS. Taken together, these results show for the first time robust RNAi within oligodendrocytes in vivo and demonstrate the important potential of siRNAs in the treatment of CNS disorders involving oligodendrocyte pathology.

Small molecule drugs are relatively effective in working on 'drugable' targets such as GPCRs, ion channels, kinases, proteases, etc but ineffective at blocking protein-protein interactions that represent an emerging class of 'nondrugable' central nervous system (CNS) targets. This article provides an overview of novel therapeutic modalities such as biologics (in particular antibodies) and emerging oligonucleotide therapeutics such as antisense, small-interfering RNA, and aptamers. Their key properties, overall strengths and limitations, and their utility as tools for target validation are presented. In addition, issues with regard to CNS targets as it relates to the blood-brain barrier penetration are discussed. Finally, examples of their application as therapeutics for the treatment of pain and some neurological disorders such as Alzheimer's disease, multiple sclerosis, Huntington's disease, and Parkinson's disease are provided.

Most cases of the dominantly inherited movement disorder, early onset torsion dystonia (DYT1) are caused by a mutant form of torsinA lacking a glutamic acid residue in the C-terminal region (torsinADeltaE). TorsinA is an AAA+ protein located predominantly in the lumen of the endoplasmic reticulum (ER) and nuclear envelope apparently involved in membrane structure/movement and processing of proteins through the secretory pathway. A reporter protein Gaussia luciferase (Gluc) shows a reduced rate of secretion in primary fibroblasts from DYT1 patients expressing endogenous levels of torsinA and torsinADeltaE when compared with control fibroblasts expressing only torsinA. In this study, small interfering RNA (siRNA) oligonucleotides were identified, which downregulate the levels of torsinA or torsinADeltaE mRNA and protein by over 65% following transfection. Transfection of siRNA for torsinA message in control fibroblasts expressing Gluc reduced levels of luciferase secretion compared with the same cells non-transfected or transfected with a non-specific siRNA. Transfection of siRNA selectively inhibiting torsinADeltaE message in DYT fibroblasts increased luciferase secretion when compared with cells non-transfected or transfected with a non-specific siRNA. Further, transduction of DYT1 cells with a lentivirus vector expressing torsinA, but not torsinB, also increased secretion. These studies are consistent with a role for torsinA as an ER chaperone affecting processing of proteins through the secretory pathway and indicate that torsinADeltaE acts to inhibit this torsinA activity. The ability of allele-specific siRNA for torsinADeltaE to normalize secretory function in DYT1 patient cells supports its potential role as a therapeutic agent in early onset torsion dystonia.

The safe and effective delivery of RNA interference (RNAi) therapeutics remains an important challenge for clinical development. The diversity of current delivery materials remains limited, in part because of their slow, multi-step syntheses. Here we describe a new class of lipid-like delivery molecules, termed lipidoids, as delivery agents for RNAi therapeutics. Chemical methods were developed to allow the rapid synthesis of a large library of over 1,200 structurally diverse lipidoids. From this library, we identified lipidoids that facilitate high levels of specific silencing of endogenous gene transcripts when formulated with either double-stranded small interfering RNA (siRNA) or single-stranded antisense 2'-O-methyl (2'-OMe) oligoribonucleotides targeting microRNA (miRNA). The safety and efficacy of lipidoids were evaluated in three animal models: mice, rats and nonhuman primates. The studies reported here suggest that these materials may have broad utility for both local and systemic delivery of RNA therapeutics.

Dorsal root injury results in substantial and often irreversible loss of sensory functions as a result of the limited regenerative capacity of sensory axons and the inhibitory barriers that prevent both axonal entry into and regeneration in the spinal cord. Here, we describe previously unknown effects of the growth factor artemin after crush injury of the dorsal spinal nerve roots in rats. Artemin not only promoted re-entry of multiple classes of sensory fibers into the spinal cord and re-establishment of synaptic function and simple behavior, but it also, surprisingly, promoted the recovery of complex behavior. These effects occurred after a 2-week schedule of intermittent, systemic administration of artemin and persisted for at least 6 months following treatment, suggesting a substantial translational advantage. Systemic artemin administration produced essentially complete and persistent restoration of nociceptive and sensorimotor functions, and could represent a promising therapy that may effectively promote sensory neuronal regeneration and functional recovery after injury.

The PATZ1 gene encodes a transcription factor that belongs to the BTB/POZ group of transcriptional regulators and has been implicated as a transcriptional repressor. We cloned cDNA from glioma cell lines and found they expressed transcript variant 2 of PATZ1. We designed a specific siRNA against PATZ1 and showed that this siRNA, but not a control randomized siRNA, reduced PATZ1 expression in glioma cells as determined by quantitative PCR. In a panel of human glioma cell lines incubated with proapoptotic FasL, those transfected with PATZ1 siRNA displayed reduced cell numbers by the MTT colorimetric assay, relative to those transfected with randomized siRNA. Further studies showed that in 10-08-MG, U-251MG, U-87MG, and T98G cells PATZ1 siRNA significantly increased apoptosis in response to incubation with soluble FasL, as shown by a morphologic acridine orange/ethidium bromide apoptotic assay. Using an apoptosis specific cDNA microarray we further demonstrated that down-regulation of PATZ1 by siRNA resulted in the upregulation of death receptor pro-apoptotic genes including caspase 8 and Death Receptor 5 (DR5) in U-373MG cells. Since DR5 is the receptor for TRAIL we tested whether PATZ1 downregulation also sensitized cells to TRAIL-induced apoptosis and found that PATZ1 siRNA, but not control siRNA, sensitized U-251MG and T98G glioma cells to TRAIL-induced apoptosis. Altogether, these data demonstrate a previously unknown role for the transcription factor PATZ1 in conferring resistance to apoptosis and indicate that modulation of PATZ1 expression may be a therapeutic strategy for gliomas.

Huntington's disease (HD) is a neurodegenerative disorder caused by expansion of a CAG repeat in the huntingtin (Htt) gene. HD is autosomal dominant and, in theory, amenable to therapeutic RNA silencing. We introduced cholesterol-conjugated small interfering RNA duplexes (cc-siRNA) targeting human Htt mRNA (siRNA-Htt) into mouse striata that also received adeno-associated virus containing either expanded (100 CAG) or wild-type (18 CAG) Htt cDNA encoding huntingtin (Htt) 1-400. Adeno-associated virus delivery to striatum and overlying cortex of the mutant Htt gene, but not the wild type, produced neuropathology and motor deficits. Treatment with cc-siRNA-Htt in mice with mutant Htt prolonged survival of striatal neurons, reduced neuropil aggregates, diminished inclusion size, and lowered the frequency of clasping and footslips on balance beam. cc-siRNA-Htt was designed to target human wild-type and mutant Htt and decreased levels of both in the striatum. Our findings indicate that a single administration into the adult striatum of an siRNA targeting Htt can silence mutant Htt, attenuate neuronal pathology, and delay the abnormal behavioral phenotype observed in a rapid-onset, viral transgenic mouse model of HD.

The rapid identification of highly specific and potent drug candidates continues to be a substantial challenge with traditional pharmaceutical approaches. Moreover, many targets have proven to be intractable to traditional small-molecule and protein approaches. Therapeutics based on RNA interference (RNAi) offer a powerful method for rapidly identifying specific and potent inhibitors of disease targets from all molecular classes. Numerous proof-of-concept studies in animal models of human disease demonstrate the broad potential application of RNAi therapeutics. The major challenge for successful drug development is identifying delivery strategies that can be translated to the clinic. With advances in this area and the commencement of multiple clinical trials with RNAi therapeutic candidates, a transformation in modern medicine may soon be realized.

During the past decade, numerous molecular mediators of neurodegenerative diseases and neurological disorders have been identified and validated, yet few novel therapies have emerged and the unmet medical needs remain high. These molecular mediators belong to target classes such as ion channels, neurotransmitters and neurotransmitter receptors, cytokines, growth factors, enzymes and other proteins. In some cases, substantial pre-clinical validation exists, but the molecular target has not been readily druggable with small molecules, proteins or antibodies. RNA interference represents a therapeutic approach applicable to such non-druggable targets. Both non-viral and viral delivery strategies are being undertaken for in vivo silencing of molecular targets by RNA interference, which has resulted in robust efficacy in animal models of Alzheimer's disease, ALS, Huntington's disease, spinocerebellar ataxia, anxiety, depression, neuropathic pain, encephalitis and glioblastoma. These proof-of-concept data in animal models, together with the commencement of clinical trials using RNA interference for macular degeneration and respiratory syncytial virus infection, point to the potential of direct RNA interference for neurological disorders and neurodegenerative diseases.

The neurotrophic growth factor artemin binds selectively to GDNF family receptor alpha3 (GFRalpha3), forming a molecular complex with the co-receptor RET which mediates downstream signaling. This signaling pathway has been demonstrated to play an important role in the survival and maintenance of nociceptive sensory neurons and in the development of sympathetic neurons. However, the presence and potential role of this artemin-responsive pathway in non-neural tissues has not been fully explored to-date. To study the distribution of GFRalpha3 and RET in adult rat and human non-neural tissues, we carried out a comprehensive immunohistochemical study. We stained major organs from the digestive, urinary, reproductive, immune, respiratory and endocrine systems, and from other systems (cardiovascular, skeletal muscle), as well as regions of the nervous system for comparison. In both rat and human, the majority of non-neural cells did not exhibit detectable GFRalpha3-like immunoreactivity. In the rat, GFRalpha3- and RET-like staining were found in the same non-neural cell type only in kidney. In the human digestive and reproductive systems, a subset of epithelial cells exhibited GFRalpha3- and RET-like staining, suggesting co-localization. In other tissues, sub-populations of cells expressed either GFRalpha3- or RET-like immunoreactivity. The functional consequences of GFRalpha3 expression in non-neural cells remain to be determined.

To clarify whether glial cell line-derived neurotrophic factor (GDNF) receptor alpha-1 (GFRalpha1), the glycosylphosphatidylinositol (GPI)-linked coreceptor for GDNF, is also a functional coreceptor for artemin (ART), we have studied receptor binding, signaling, and neuronal survival. In cell-free binding studies, GFRalpha1-Ig displayed strong preferential binding to GDNF, though in the presence of soluble RET, weak binding to ART could also be detected. However, using GFRalpha1-transfected NB41A3 cells, ART showed no detectable competition against the binding of (125)I-labeled GDNF. Moreover, ART failed to induce phosphorylation of extracellular signal-related kinase (ERK) and Akt in these cells and was >10(4)-fold less potent than GDNF in stimulating RET phosphorylation. When rat primary dorsal root ganglion (DRG) neurons were used, only the survival promoting activity of GDNF and not that of ART was blocked by an anti-GFRalpha1 antibody. These results indicate that although ART can interact weakly with soluble GFRalpha1 constructs under certain circumstances in vitro, in cell-based functional assays GFRalpha1 is at least 10 000-fold selective for GDNF over ART. The extremely high selectivity of GFRalpha1 for GDNF over ART and the low reactivity of ART for this receptor suggest that GFRalpha1 is not likely to be a functional coreceptor for ART in vivo.

This article focuses on the GDNF family of neurotrophic factors as a potential new class of therapeutics for neuropathic pain, with a particular emphasis on the ligands, artemin and GDNF. In vivo activity of the ligands, expression of ligands and receptors after peripheral nerve injury, and modulation of nerve injury-induced changes by the ligands are reviewed in detail. Structural considerations, particularly with regard to implications for binding interactions and biological activity are discussed.

The growth of injured axons in the adult mammalian CNS is limited after injury. Three myelin proteins, Nogo, MAG (myelin-associated glycoprotein), and OMgp (oligodendrocyte myelin glycoprotein), bind to the Nogo-66 receptor (NgR) and inhibit axonal growth in vitro. Transgenic or viral blockade of NgR function allows axonal sprouting in vivo. Here, we administered the soluble function-blocking NgR ectodomain [aa 27-310; NgR(310)ecto] to spinal-injured rats. Purified NgR(310)ecto-Fc protein was delivered intrathecally after midthoracic dorsal over-hemisection. Axonal sprouting of corticospinal and raphespinal fibers in NgR(310)ecto-Fc-treated animals correlates with improved spinal cord electrical conduction and improved locomotion. The ability of soluble NgR(310)ecto to promote axon growth and locomotor recovery demonstrates a therapeutic potential for NgR antagonism in traumatic spinal cord injury.

The Nogo66 receptor (NgR1) is a neuronal, leucine-rich repeat (LRR) protein that binds three central nervous system (CNS) myelin proteins, Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein, and mediates their inhibitory effects on neurite growth. Although the LRR domains on NgR1 are necessary for binding to the myelin proteins, the exact epitope(s) involved in ligand binding is unclear. Here we report the generation and detailed characterization of an anti-NgR1 monoclonal antibody, 7E11. The 7E11 monoclonal antibody blocks Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein binding to NgR1 with IC50 values of 120, 14, and 4.5 nm, respectively, and effectively promotes neurite outgrowth of P3 rat dorsal root ganglia neurons cultured on a CNS myelin substrate. Further, we have defined the molecular epitope of 7E11 to be DNAQLR located in the third LRR domain of rat NgR1. Our data demonstrate that anti-NgR1 antibodies recognizing this epitope, such as 7E11, can neutralize CNS myelin-dependent inhibition of neurite outgrowth. Thus, specific anti-NgR1 antibodies may represent a useful therapeutic approach for promoting CNS repair after injury.

The clinical management of neuropathic pain is particularly challenging. Current therapies for neuropathic pain modulate nerve impulse propagation or synaptic transmission; these therapies are of limited benefit and have undesirable side effects. Injuries to peripheral nerves result in a host of pathophysiological changes associated with the sustained expression of abnormal pain. Here we show that systemic, intermittent administration of artemin produces dose- and time-related reversal of nerve injury-induced pain behavior, together with partial to complete normalization of multiple morphological and neurochemical features of the injury state. These effects of artemin were sustained for at least 28 days. Higher doses of artemin than those completely reversing experimental neuropathic pain did not elicit sensory or motor abnormalities. Our results indicate that the behavioral symptoms of neuropathic pain states can be treated successfully, and that partial to complete reversal of associated morphological and neurochemical changes is achievable with artemin.

Neuropathic pain is a chronic condition that is caused by injury to the nervous system. Unlike acute pain, which is protective, neuropathic pain persists and serves no useful purpose, and severely affects quality of life. However, present therapies have modest efficacy in most patients, are palliative rather than curative, and their side effects represent significant limitations. Tremendous progress has been made over the past decade in our understanding of the biology of pain sensory neurons. The recent discovery that neurotrophic factors play an important role in neuropathic pain indicates that these pathways could serve as novel intervention points for therapy. Moreover, neurotrophic factors have the potential to address the underlying pathophysiology of neuropathic pain, thereby halting or reversing the disease process.

Activation of the RET receptor tyrosine kinase by glial-derived neurotrophic factor family members is dependent on a family of coreceptors, GFRalpha1-4. GFRalpha3 preferentially binds the newest member of the glial-derived neurotrophic factor family of ligands, artemin. The major site of GFRalpha3 expression is in the dorsal root ganglion; however, the class of sensory neurons that expresses GFRalpha3 has not been reported previously. Using immunohistochemical methods, we show that the majority of dorsal root ganglion cells that express GFRalpha3 also express vanilloid receptor type 1, peripherin, RET, trkA and calcitonin gene-related peptide. In addition, a significant subpopulation of GFRalpha3-expressing cells also binds the lectin IB4. We demonstrate that GFRalpha3 artemin neurons are immunopositive for markers expected of nociceptors and include a subset of neurons distinct from the GDNF-responsive population. Our results indicate artemin may exert selective effects on pain sensation.

Human motoneuron cell lines will be valuable tools for spinal cord research and drug discovery. To create such cell lines, we immortalized NCAM(+)/neurofilament(+) precursors from human embryonic spinal cord with a tetracycline repressible v-myc oncogene. Clonal NCAM(+)/neurofilament(+) cell lines differentiated exclusively into neurons within 1 week. These neurons displayed extensive processes, exhibited immunoreactivity for mature neuron-specific markers such as tau and synaptophysin, and fired action potentials upon current injection. Moreover, a clonal precursor cell line gave rise to multiple types of spinal cord neurons, including ChAT(+)/Lhx3(+)/Lhx4(+) motoneurons and GABA(+) interneurons. These neuronal restricted precursor cell lines will expedite the elucidation of molecular mechanisms that regulate the differentiation, maturation and survival of specific subsets of spinal cord neurons, and the identification and validation of novel drug targets for motoneuron diseases and spinal cord injury., (Copyright 2000 Wiley-Liss, Inc.)

A renewable source of human sensory neurons would greatly facilitate basic research and drug development. We had established previously conditionally immortalized human CNS cell lines that can differentiate into functional neurons (). We report here the development of an immortalized human dorsal root ganglion (DRG) clonal cell line, HD10.6, with a tetracycline-regulatable v-myc oncogene. In the proliferative condition, HD10.6 cells have a doubling time of 1.2 d and exhibit a neuronal precursor morphology. After differentiation of clone HD10.6 for 7 d in the presence of tetracycline, v-myc expression was suppressed, and >50% of the cells exhibited typical neuronal morphology, stained positively for neuronal cytoskeletal markers, and fired action potentials in response to current injection. Furthermore, this cell line was fate-restricted to a neuronal phenotype; even in culture conditions that promote Schwann cell or smooth muscle differentiation of neural crest stem cells, HD10.6 differentiated exclusively into neurons. Moreover, differentiated HD10.6 cells expressed sensory neuron-associated transcription factors and exhibited capsaicin sensitivity. Taken together, these data indicate that we have established an immortalized human DRG cell line that can differentiate into sensory neurons with nociceptive properties. The cell line HD10.6 represents the first example of a human sensory neuronal line and will be valuable for basic research, as well as for the discovery of novel drug targets and clinical candidates.

Human central nervous system (CNS) cell lines would substantially facilitate drug discovery and basic research by providing a readily renewable source of human neurons. We isolated clonal human CNS cell lines that had been immortalized with a tetracycline (Tc)-responsive v-myc oncogene; addition of Tc to the growth medium suppressed the oncoprotein rapidly and virtually completely, allowing differentiation to proceed. Two classes of bipotent precursor cells were immortalized: the first class had a default differentiation pathway of neurons only, and the second class had a default differentiation pathway of neurons and astrocytes. We found that after exposure to different external signals in vitro, the environment is capable of redirecting the fate of a particular cell, even in the case of the bipotent precursor cell whose default differentiation pathway was neurons only. These data suggest that extrinsic cues can prevail over intrinsic determinants in directing cell fate in the human CNS.

Growth medium and substrate regulate the proliferation and differentiation of neural progenitor cells in vitro and the expression of cell type-specific histochemical markers. An important question is whether neural progenitor cells exhibit voltage- and ligand-gated currents, features characteristic of neurons, and whether these currents are regulated differentially by growth conditions. Another issue of interest is whether passaged progenitor cells, after expansion with basic fibroblast growth factor (FGF-2), exhibit the same degree of plasticity as their primary counterparts, or whether they are more committed to a particular phenotype. In primary cultures of embryonic rat hippocampal progenitor cells, growth in proliferative conditions (FGF-2) was associated with low levels of sodium, calcium, N-methyl-D-aspartate (NMDA), and kainate currents compared with other growth conditions. After multiple passages in the continued presence of FGF-2, sodium, calcium, and NMDA, responses declined further; interestingly, kainate and gamma-aminobutyric acid (GABA) responses remained substantial. Moreover, the expression of functional channels and receptors in primary cultures of progenitor cells is up-regulated strongly by growth factors such as BDNF, and NT-3, whereas sodium and calcium currents in passaged cultures respond to such growth conditions to a lesser extent. Kainate and GABA responses were present to a significant extent in passaged cultures, independent of growth condition. We conclude that environmental cues regulate different channels and receptors in distinct ways in neural progenitor cells.

A regulatable retroviral vector in which the v-myc oncogene is driven by a tetracycline-controlled transactivator and a human cytomegalovirus minimal promoter fused to a tet operator sequence was used for conditional immortalization of adult rat neuronal progenitor cells. A single clone, HC2S2, was isolated and characterized. Two days after the addition of tetracycline, the HC2S2 cells stopped proliferating, began to extend neurites, and expressed the neuronal markers tau, NeuN, neurofilament 200 kDa, and glutamic acid decarboxylase in accordance with the reduced production of the v-myc oncoprotein. Differentiated HC2S2 cells expressed large sodium and calcium currents and could fire regenerative action potentials. These results suggest that the suppression of the v-myc oncogene may be sufficient to make proliferating cells exit from cell cycles and induce terminal differentiation. The HC2S2 cells will be valuable for studying the differentiation process of neurons.

1. The functional properties of sodium, potassium, calcium, N-methyl-D-aspartate (NMDA), kainate, and gamma-aminobutyric acid (GABA) currents were studied in dissociated monolayer cultures of fetal human brain neurons, using the whole cell patch-clamp technique. 2. Sodium currents were characterized with respect to the following properties: current density, voltage dependence of activation, voltage dependence of inactivation, and sensitivity to tetrodotoxin (TTX). All sodium currents exhibited voltage dependencies of activation and inactivation, and sensitivities to TTX that are characteristic of the neuronal form of the sodium current. 3. At least two types of potassium current were present, resembling the delayed rectifier and fast-inactivating potassium current. These two types of potassium current were distinguishable by their different kinetics, voltage dependencies of activation and inactivation, and sensitivities to 4-aminopyridine and tetraethylammonium. 4. High-voltage-activated calcium channel currents were present and were characterized with respect to current density, voltage dependencies of activation and inactivation, and sensitivity to cadmium. Low-voltage-activated calcium channel currents were also present. 5. NMDA- and kainate-gated currents were studied with respect to current density, time course, and current-voltage relationship. Kainate currents were also characterized with respect to inhibition by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In addition, NMDA and kainate responses were compared for cortical versus cerebellar neurons. NMDA responses, which are only found in neurons, were present, confirming the neuronal phenotype suggested by the presence of the neuronal form of the sodium current. Nondesensitizing kainate currents were also present, with a half-maximally effective concentration (EC50) of approximately 200 microM for kainate; CNQX inhibited the kainate current with a half-inactivating concentration of 0.55 microM. 6. GABA-gated currents were characterized with respect to current density, time course, receptor subtype, desensitization, dose response, current-voltage relationship, ionic selectivity, pharmacology, and potentiation by the neurosteroid 5 alpha-pregnan-3 alpha-ol-11,20-dione (alfaxalone). Desensitizing GABAA currents were selective for chloride, inhibited by bicuculline and tert-butyl-bicyclophosphorothionate, and potentiated by diazepam, pentobarbital sodium, and alfaxalone. The EC50 for GABA was 15 microM.

P-type Ca2+ channels in cerebellar Purkinje neurons and N-type Ca2+ channels in sympathetic neurons were found to be inhibited by D2 dopamine receptor antagonists with diverse structures, including phenothiazines (chlorpromazine and thioridazine), diphenylbutylpiperidines (fluspirilene and pimozide), butyrophenones (haloperidol and spiperone), and a piperazine (fluphenazine). Dopamine and quinpirole had no effect on P-type Ca2+ channels. In all cases, inhibition was characterized by slow onset and offset. The effects of P-type and N-type channels were very similar. Fluspirilene was the most potent of the drugs, with EC50 values of 6 microM for P-type current and 2 microM for N-type current. Block of P-type channels by fluspirilene was voltage dependent, being enhanced by depolarized holding potentials, and use dependent, being enhanced by higher stimulation frequencies. The effect of fluspirilene on the P-type Ca2+ channel current was not prevented by simultaneous exposure to the peptide toxin omega-agatoxin IVA, indicating that fluspirilene binds to a distinct site on the channel. The results suggest that N-type and P-type Ca2+ channels possess similar binding sites for dopamine receptor antagonists and that block of N-type and P-type channels is relatively weak, compared with that of some T-type and L-type Ca2+ channels.

Block of Ca2+ channel current by dihydropyridines and by omega-conotoxin (omega-CgTx) was studied in a variety of freshly dissociated rat neurons. In most neurons, including those from dorsal root ganglia, sympathetic ganglia, spinal cord, cerebral cortex, and hippocampus, nitrendipine and omega-CgTx each blocked a fraction of the high-threshold current, but a substantial fraction of current remained even when the two blockers were applied together at saturating concentrations. An extreme case was cerebellar Purkinje neurons, in which very little current was blocked by either nitrendipine or omega-CgTx. These results demonstrate the existence in mammalian neurons of high-threshold channels that are resistant to both omega-CgTx and dihydropyridine blockers. Such channels might underlie instances of synaptic transmission and other processes that depend on Ca2+ entry but are not sensitive to these blockers.

An intriguing question regarding neuronal development is how neurons choose which neurotransmitter and/or peptide to express among over 40 candidates. We find that heart cell conditioned medium (CM) induces a number of neuropeptides and/or their precursor mRNAs, as well as acetylcholine, in cultured rat sympathetic neurons: substance P, somatostatin, vasoactive intestinal polypeptide, enkephalin derivatives, and cholecystokinin, but not neuropeptide Y. Different patterns of peptide induction were observed for CMs from primary cultures of heart, gut, and skin. Acetylcholine and substance P were induced most effectively by serum-free heart cell CM; enkephalin derivatives were induced most effectively by skin cell CM; and somatostatin and vasoactive intestinal polypeptide were induced equally well by all of the CMs. These observations suggest the possibility that many distinct, diffusible factors can influence the choice of transmitter and/or peptide phenotype in developing neurons.

The effects of a recently identified blocker of neuronal nicotinic transmission, toxin F, were studied in cultured sympathetic neurons. Single principal neurons, dissociated from superior cervical ganglia of newborn rats, were grown on cardiac myocytes in microculture. The toxin blocked nicotinic synaptic potentials in these cultures but had no effect on muscarinic interactions. When toxin F was applied by addition to the perfusion medium, the concentration required for blocking most of the nicotinic potential was 40 nM, and the recovery from blockade was slow (t1/2 = 95 +/- 64 min). When the toxin was briefly applied by pressure ejection from a pipette, the concentration in the pipette necessary for blockade was 21 microM, and 30-60% of the response recovered within a few minutes while the remainder recovered slowly (t1/2 of the remainder = 105 +/- 82 min). One possible explanation for the difference in recovery time is that toxin F binds initially with low affinity to the nicotinic receptor but with time the toxin receptor complex converts to a high affinity state. The presence of dihydro-beta-erythroidine during perfusion of toxin F prevented the long-lasting blockade by the toxin, suggesting that toxin F and dihydro-beta-erythroidine act through a common binding site. The specificity, potency, and slow reversibility of the effects of toxin F make it useful as a probe for studying neuronal nicotinic receptors of cultured sympathetic neurons.

The regrowth of sensory axons into the spinal cord of juvenile bullfrogs was studied after disruption of these fibers in the dorsal root. Within 9 d after the root had been frozen, regenerating sensory axons had reached the spinal cord, as revealed by labeling with horseradish peroxidase. Growth into the spinal cord, however, was much slower. Even several months after denervation, very few fibers had reestablished any of their normal longitudinal projections within the dorsal funiculus. Eventually, however, sensory axons grew across the region and into the dorsal horn. Intracellular recordings from motoneurons revealed that these axons made functional reconnections with spinal neurons. Muscle sensory axons established direct, monosynaptic inputs to motoneurons, whereas cutaneous fibers innervated these neurons polysynaptically. Moreover, sensory afferents from a particular muscle distinguished among different classes of motoneurons, just as in normal frogs. Thus, specific synaptic pathways can be reestablished by regenerating sensory axons if they can reach their appropriate target region within the spinal cord.

The distribution of putative nicotinic receptors on cultured neurons from the rat superior cervical ganglion was determined by electron microscopic autoradiography using a radioactively labeled snake venom neurotoxin, toxin F. In a previous study, we demonstrated that toxin F blocks nicotinic transmission in these cultures of sympathetic neurons and in intact superior cervical ganglia. [125I]toxin F bound to two sites in these cultures: one site that was also recognized by the neuromuscular blocker, alpha-bungarotoxin, and a second site that was not. Since alpha-bungarotoxin neither blocks nicotinic transmission nor prevents the blocking effects of toxin F, the site specific to the binding of toxin F most probably represents neuronal nicotinic receptors. The total number of each of the toxin F binding sites was unaffected by culture conditions that are known to influence the extent to which these sympathetic neurons synthesize norepinephrine or acetylcholine. Autoradiographic analysis performed under saturating binding conditions (80 nM [125I]toxin F) revealed that the density of [125I]toxin F binding at synaptic membranes was about 5000 sites/micron 2, either in the absence of any competing ligand or in the presence of 2 microM alpha-bungarotoxin. In the presence of 2 microM unlabeled toxin F, there was no detectable binding at synapses. The density of these toxin F-specific sites was at least 80-fold higher at synaptic membranes than elsewhere. On the other hand, the data suggest that the toxin F binding site shared with alpha-bungarotoxin is exclusively extrasynaptic. Two micromolar alpha-bungarotoxin decreased the density of [125I]toxin F binding at non-synaptic sites by approximately two-thirds. These experiments support the hypothesis that toxin F blocks cholinergic transmission in cultures of sympathetic neurons by binding to nicotinic receptors and suggests that these receptors are highly clustered at synaptic membranes.