Comparative evaluation of the potency and antigenicity of two distinct BoNT/A-derived formulations.

IncobotulinumtoxinA (Xeomin®) and onabotulinumtoxinA (BOTOX®) are unique botulinum neurotoxin type A (BoNT/A)-derived drugs. IncobotulinumtoxinA utilizes the naked 150 kDa holotoxin portion of BoNT/A, whereas onabotulinumtoxinA uses the complete native 900 kDa complex as drug substance. On the basis of purportedly similar pharmacological characteristics, these formulations were evaluated for potency by LD50 and mouse Digit Abduction Score (DAS) bioassays. DAS was also used to assess antigenicity. Full-range DAS dose-response profiles were achieved with four lots of each product, with similar observations between lots for a given product. Between products, however, the mean DAS potency of incobotulinumtoxinA (ED50 range 7.0-10.2 U/kg) was significantly lower than that of onabotulinumtoxinA (ED50 range 4.4-6.4 U/kg), consistent with lower measured potencies in the LD50 assay for incobotulinumtoxinA (potency range 62-82 U). In assessments of DAS duration of effect at similar unit doses, the observed lower potency of incobotulinumtoxinA translated into decreased peak efficacy and dose effect over time (i.e. shorter duration). In contrast, at equi-efficacious doses yielding near-maximal DAS responses, both toxin formulations were uniformly inhibited in a statistically significant manner when preincubated with rabbit-derived, onabotulinumtoxinA-neutralizing antibodies, supporting the position that inhibition of 150 kDa holotoxin serves as the common basis for neutralization and, therefore, incobotulinumtoxinA would not be expected to be effective in onabotulinumtoxinA-immunoresistant subjects (and vice versa). Further, with lower lot-to-lot relative potency, incobotulinumtoxinA is not dose-equivalent or interchangeable with onabotulinumtoxinA, suggesting that various aspects of drug product formulation may influence observed pharmacology.

Normal apoptosis levels in mice expressing one alpha7 nicotinic receptor null and one L250T mutant allele.

The alpha7 nicotinic receptor (nAChR) is a ligand-gated ion channel mediating cholinergic transmission throughout the nervous system. To further characterize the function of this receptor, we generated mice expressing the alpha7 L250T nAChR mutation and demonstrated that homozygous (T/T) L250T mice die within 24 h of birth and display extensive apoptosis and abnormal layering within their cortex. We now demonstrate that mice with one alpha7 null and one L250T allele (-/T) show little apoptosis and normal development of their cortex yet exhibit the same lethal phenotype as T/T mice. Furthermore, L250T mice show normal levels of apoptosis in other nervous system regions expressing alpha7 nAChRs. These results suggest that apoptosis is not the cause of death for L250T neonatal mice.

Synaptogenesis and myopathy under acetylcholinesterase overexpression.

Environmental, congenital, and acquired immunological insults perturbing neuromuscular junction (NMJ) activity may induce a variety of debilitating neuromuscular pathologies. However, the molecular elements linking NMJ dysfunction to long-term myopathies are unknown. Here, we report dramatically elevated levels of mRNA encoding c-Fos and the "readthrough" (R) variant of acetylcholinesterase (AChE) in muscles of transgenic mice overexpressing synaptic (S) AChE in motoneurons and in control mice treated with the irreversible cholinesterase inhibitor diisopropylfluorophosphonate (DFP). Tongue muscles from DFP-treated and AChE-S transgenic mice displayed exaggerated neurite branching and disorganized, wasting fibers. Moreover, diaphragm muscles from both transgenic and DFP-treated mice exhibited NMJ proliferation. 2'-O-methyl-protected antisense oligonucleotides targeted to AChE mRNA suppressed feedback upregulation of AChE and ameliorated DFP-induced NMJ proliferation. Our findings demonstrate common transcriptional responses to cholinergic NMJ stress of diverse origin, and implicate deregulated AChE expression in excessive neurite outgrowth, uncontrolled synaptogenesis, and myopathology.

Mice homozygous for the L250T mutation in the alpha7 nicotinic acetylcholine receptor show increased neuronal apoptosis and die within 1 day of birth.

The alpha7 nicotinic acetylcholine receptor (nAChR) has been implicated in modulating neurotransmitter release and may play a role in the regulation of neuronal growth and differentiation. A threonine for leucine 247 substitution in the channel domain of the chick alpha7 nAChR increases agonist affinity and decreases the rate of desensitization, creating a "gain of function" model for this receptor. We have generated mice that express the analogous mutation (L250T) in the alpha7 nAChR using the techniques of homologous recombination and here report their characteristics. Mice heterozygous (+/T) for the L250T mutation are viable, fertile, and anatomically normal compared with wild-type littermates. In contrast, homozygous (T/T) L250T mice die within 2-24 h of birth. Brains of T/T mouse pups exhibit a marked reduction in alpha7 nAChR protein levels and show extensive apoptotic cell death throughout the somatosensory cortex. Furthermore, alpha7 L250T nAChRs are functionally expressed on neurons within the brains of T/T neonatal mice and have properties that are consistent with those observed for the rat alpha7 L250T and the chick alpha7 L247T mutant nAChRs expressed in oocytes. These findings indicate that neurons in the developing brain expressing only alpha7 L250T mutant nAChRs are susceptible to abnormal apoptosis, possibly due to increased Ca2+ influx.

The alpha7 nicotinic acetylcholine receptor in neuronal plasticity.

A growing body of evidence indicates that neuronal nicotinic acetylcholine receptors (nAChRs), in addition to promoting fast cholinergic transmission, may modulate other neuronal activities within the central nervous system (CNS). In particular, the alpha7 nAChR is highly permeable to Ca2+ and may serve a distinct role in regulating neuronal plasticity. By elevating intracellular Ca2+ levels in discrete neuronal locations, these ligand-gated ion channels may influence numerous physiological processes in developing and adult CNS. In this article, we review evidence that both pre- and postsynaptic alpha7 nAChRs modulate transmitter release in the brain and periphery through Ca2+-dependent mechanisms. The possible role of alpha7 nAChRs in regulating neuronal growth and differentiation in developing CNS is also evaluated. We consider an interaction between cholinergic and glutamatergic transmission and propose a hypothesis on the possible coregulation of intracellular Ca2+ by N-methyl-D-aspartate (NMDA) receptors and alpha7 nAChRs. Finally, the clinical significance of alterations in the normal function of alpha7 nAChRs is discussed as it pertains to prenatal nicotine exposure, schizophrenia, and epilepsy.

Manipulations of ACHE gene expression suggest non-catalytic involvement of acetylcholinesterase in the functioning of mammalian photoreceptors but not in retinal degeneration.

To explore role(s) of acetylcholinesterase (AChE) in functioning and diseased photoreceptors, we studied normal (rd/+) and degenerating (rd/rd) murine retinas. All retinal neurons, expressed AChEmRNA throughout fetal development. AChE and c-Fos mRNAs peaked at post-natal days 10-12, when apoptosis of rd/rd photoreceptors begins. Moreover, c-Fos and AChEmRNA were co-overexpressed in rd/rd mice producing transgenic human (h), and host (m) AChE, but not in rd/+ mice. However, mAChE overexpression also occurred in transgenics expressing human serum albumin. Drastic variations in AChE catalytic activity were ineffective during development. Neither transgenic excess nor diisopropylfluorophosphonate (DFP) inhibition (80%) affected the rd phenotype; nor did DFP exposure induce photoreceptor degeneration or affect other key cholinergic proteins in rd/+ mice, unlike reports of adult mice and despite massive induction under DFP of c-Fos70 years). Therefore, the extreme retinal sensitivity to AChE modulation may reflect non-catalytic function(s) of AChE in adult photoreceptors. These findings exclude AChE as causing the rd phenotype, suggest that its primary function(s) in mammalian retinal development are non-catalytic ones and indicate special role(s) for the AChE protein in adult photoreceptors.

Highly sensitive radioactive in situ hybridization using full length hydrolyzed riboprobes to detect alpha 2 adrenoceptor subtype mRNAs in adult and developing rat brain.

The mRNA expression of highly homologous alpha 2 adrenoceptor subtypes was determined using a highly sensitive in situ hybridization protocol that allowed the detection of low abundance mRNA. Full-length 35S-labeled riboprobes specific for alpha 2A, alpha 2B and alpha 2C adrenoceptors were used for maximal sensitivity. The probes were hydrolyzed to an average length of 600 bp which, in combination with proteinase K digestion, resulted in optimal probe penetration in developmental and adult tissue. The expression intensity could be quantified and the ontogeny of receptor mRNA expression determined. At the same time receptor binding sites or functional proteins could be detected simultaneously in adjacent sections, because fresh frozen and post-fixed tissue was used.

Calcium regulation of agonist binding to alpha7-type nicotinic acetylcholine receptors in adult and fetal rat hippocampus.

Quantitative autoradiography was used to compare the binding properties of alpha7-type nicotinic acetylcholine receptors in fetal and adult rat hippocampus. Whereas there were high levels of 125I-alpha-bungarotoxin (125I-alpha-BTX) binding throughout fetal hippocampal field CA1, there was a significant decrease in binding site density in the adult. The affinity of 125I-alpha-BTX binding, as well as alpha-cobratoxin and nicotine potency to displace 125I-alpha-BTX, did not change with age. Addition of Ca2+ to the assay buffer did not alter 125I-alpha-BTX binding, or alpha-cobratoxin inhibition of 125I-alpha-BTX binding, although it significantly increased nicotine affinity at both ages. The effect of Ca2+ on agonist affinity was dose-dependent, with an EC50 value of 0.25-0.5 mM. Ca2+ also significantly increased the cooperativity of nicotine displacement curves in stratum oriens of the adult, but not in the fetus. These findings indicate that the properties of hippocampal 125I-alpha-BTX binding sites are largely similar across age. Ca2+ selectively enhances the affinity of agonist binding, with no change in antagonist binding. This ionic effect may result from potentiation of agonist binding to a desensitized state of the alpha7 nicotinic acetylcholine receptor and may represent an important neuroprotective mechanism.

Genetic manipulations of cholinergic communication reveal trans-acting control mechanisms over acetylcholine receptors.

Several approaches have been developed for genetic modulations of receptor expression. These initiated with gene cloning and heterologous expression in microinjected Xenopus oocytes, and proceeded through transgenic expression and genomic disruption of receptor genes in mice. In addition, antisense treatments have reduced receptor levels in a transient, reversible manner. Integration of foreign DNA with host genomic sequences yields both cis- and trans-acting responses. These may depend on the DNA integration site, host cells condition and most importantly, the affected signal transduction circuit. For example, acetylcholinesterase (AChE) overexpression in microinjected Xenopus tadpoles has been shown to upregulate alpha-bungarotoxin binding levels, indicating trans-acting control conferring overproduction of muscle nicotinic acetylcholine receptors. In transgenic mice expressing human AChE, the hypothermic response to oxotremorine was suppressed, reflecting modified levels of brain muscarinic receptors. To dissociate the feedback processes occurring in transfected cells from responses related to DNA integration, we examined the endogenous expression of the alpha 7 neuronal nicotinic acetylcholine receptor in PC12 cells transfected with DNA vectors carrying alternative splicing variants of human AChE mRNA. Our findings demonstrate suppression of alpha 7 receptor levels associated with the accumulation of foreign DNA in the transfected cells. Acetylcholine receptor levels thus depend on multiple elements, each of which should be considered when genetic interventions are employed.

Expression of alpha 2 adrenoceptors during rat brain development--I. Alpha 2A messenger RNA expression.

The distribution of alpha 2A adrenoceptor messenger RNA expression in developing rat brain was characterized using in situ hybridization with 35S-labeled riboprobes. Intense hybridization signal was detected as early as embryonic day 14 in several areas adjacent to the forebrain and hindbrain germinal zones and in central noradrenergic neurons. A marked increase in messenger RNA expression was observed throughout the brain during late prenatal development, consistent with the migration and maturation of neurons in developing brain structures. In embryonic brain, there was a temporal and spatial correspondence in the appearance of alpha 2A messenger RNA expression and binding sites labeled with [3H]idazoxan or p-[125I]iodoclonidine, indicating translation into receptor protein at an early stage of development. Whereas the presynaptic expression remained constant throughout development, there was an early postnatal decline of alpha 2A receptor expression in many brain regions, including the olfactory bulb, cortex, caudate-putamen, hippocampus, thalamus, hypothalamus and medulla. Thereafter, messenger RNA expression increased, establishing an adult-like pattern during the second postnatal week, but remained low in areas such as the caudate-putamen, thalamus and hippocampus, which do not exhibit extensive expression in the adult. The transient perinatal expression of this alpha 2 adrenoceptor type, which coincides with a period of hyperreactivity to sensory stimuli in the locus coeruleus, may indicate a specific functional role for the alpha 2A receptor in the developing rat brain. The early and intense expression in olfactory structures suggests an involvement in early olfactory learning. The pattern of widespread, transient expression of alpha 2A receptors in the fetal brain is in marked contrast to the postnatal development of the alpha 2C receptor type.

Expression of alpha 2 adrenoceptors during rat brain development--II. Alpha 2C messenger RNA expression and [3H]rauwolscine binding.

The distributions of alpha 2C adrenoceptor messenger RNA and high-affinity [3H]rauwolscine binding sites were characterized in developing rat brain. Using in situ hybridization with 35S-labeled riboprobes directed against the third intracellular loop, alpha 2C messenger RNA expression appeared in an adult-like pattern during the first and second postnatal weeks, in the anterior olfactory nucleus, caudate-putamen, olfactory tubercles, islands of Calleja and hippocampus, following the time-course of maturation of these structures. Only in the cerebellum was alpha 2C messenger RNA transiently expressed during the critical period of granule cell development. High-affinity [3H]rauwolscine binding sites were detected using receptor autoradiography and revealed a similar spatial and temporal time-course of appearance during rat brain development. The highest numbers of binding sites were detected in the olfactory tubercles and islands of Calleja, and moderate numbers in the anterior olfactory nucleus, caudate-putamen and hippocampus. Like alpha 2C messenger RNA expression, high-affinity [3H]rauwolscine binding sites were transiently expressed in the cerebellum. In some areas (e.g., the substantia nigra), [3H]rauwolscine binding sites were detected even though alpha C2 messenger RNA expression was absent. The strong spatial and temporal correspondence between messenger RNA expression and radioligand binding supports the conclusion that [3H]rauwolscine selectively labels alpha 2C adrenoceptors in the rat brain. The developmental pattern which was observed is in marked contrast to the early, transient expression of the alpha 2A adrenoceptor. Thus, the alpha 2A and alpha 2C receptor types may serve distinct functional roles in the developing brain.

Regulation of alpha7 nicotinic acetylcholine receptors in the developing rat somatosensory cortex by thalamocortical afferents.

Distributions of alpha7 nicotinic acetylcholine receptor (nAChR) mRNA and [125]alpha-bungarotoxin (alpha-BTX) binding sites in the developing rat somatosensory cortex were characterized in relation to acetylcholinesterase (AChE) histochemical staining of thalamocortical terminals to investigate the role of this receptor in cortical development. Using quantitative in situ hybridization and receptor autoradiography, elevated levels of mRNA and binding-site expression were first detected at post-natal day 1 (P1) in deep and superficial layers, just beneath the AChE-stained thalamocortical terminals. Onset of expression occurred approximately 1 d after ingrowth of AChE-stained thalamocortical afferents. By P5, mRNA and binding-site expression exhibited a disjunctive, barrel-like pattern in layer IV and, more clearly, in layer VI. The mRNA and binding-site expressions peaked at approximately 1 week postnatal and then declined to adult levels. Unilateral electrolytic or cytochemical lesions placed in the thalamic ventrobasal complex at P0 (just as thalamocortical afferents are innervating the cortex) and at P6 (when the somatotopic map is well established) resulted in a marked reduction of alpha7 nAChR mRNA and [125]alpha-BTX binding-site levels in layers IV and VI, indicating their regulation by thalamocortical afferents. With P6 lesions, this reduction was observed as early as 6 hr postlesion. These results suggest that alpha7 nAChRs are localized primarily on cortical cells in rat somatosensory cortex and provide further evidence for thalamocortical influence on cortical ontogeny. These data also suggest a role for cholinergic systems during a critical period of cortical synaptogenesis.

Localization of alpha 7 nicotinic receptor subunit mRNA and alpha-bungarotoxin binding sites in developing mouse somatosensory thalamocortical system.

Previous studies in rat, showing a transient pattern of expression of the alpha 7 nicotinic acetylcholine receptor in the ventrobasal thalamus and barrel cortex during the first 2 postnatal weeks, suggest that these receptors may play a role in development of the thalamocortical system. In the present study, in situ hybridization and radiolabeled ligand binding were employed to examine the spatiotemporal distribution of alpha 7 mRNA and alpha-bungarotoxin binding sites in the thalamocortical pathway of mouse during early postnatal development. As in the rat, high levels of alpha 7 mRNA and alpha-bungarotoxin binding sites are present in the barrel cortex of mouse during the first postnatal week. Both alpha 7 mRNA and its receptor protein are observed in all cortical laminae, with the highest levels seen in the compact cortical plate, layer IV, and layer VI. When viewed in a tangential plane, alpha 7 mRNA and alpha-bungarotoxin binding sites delineate a whisker-related barrel pattern in layer IV by P3-5. Quantitative analysis reveals a dramatic decrease in the levels of expression of alpha 7 mRNA and alpha-bungarotoxin binding sites in the cortex by the end of the second postnatal week. Unlike in the rat, only low levels of alpha 7 mRNA or alpha-bungarotoxin binding sites are present in the ventrobasal complex of the mouse thalamus. The broad similarities between the thalamocortical development of rat and mouse taken together with the present results suggest that alpha 7 receptors located on cortical neurons, rather than on thalamic neurons, play a role in mediating aspects of thalamocortical development.

Developmental expression of alpha 7 neuronal nicotinic receptor messenger RNA in rat sensory cortex and thalamus.

The distribution of alpha 7 messenger RNA expression was characterized in developing rat cortex and thalamus. Northern blot analysis of neonatal and adult cortex revealed a single messenger RNA transcript of 5.7 kb. Using in situ hybridization with both full length and short 35S-labeled alpha 7 riboprobes, a distinct transient expression of messenger RNA within sensory cortex and thalamus, during early postnatal development, was observed. alpha 7 transcripts were expressed in low levels as early as embryonic day 13 in the ventricular zone of the neocortex, and as early as embryonic day 15 in the thalamic neuroepithelium. A marked increase in messenger RNA levels was observed during the late prenatal period in both sensory and non-sensory regions of the cortex and thalamus. Moderate to high levels of messenger RNA were maintained into the first postnatal week, followed by a decline into adulthood. alpha 7 messenger RNA expression was significantly higher in the anterodorsal, lateral dorsal, ventral posterior medial and ventral posterior lateral thalamic nuclei of postnatal day 7 pups than in adult brains. Expression of messenger RNA within dorsal lateral geniculate, ventral lateral geniculate and medial geniculate did not show a significant reduction with age. Within the developing cortex, messenger RNA expression delineated the primary somatosensory, auditory and visual cortices in a unique laminar pattern that was consistently and significantly higher than in the adult in superficial layer VI. Higher levels of expression were also observed in retrosplenial cortex at postnatal day 7 than in the adult. Tangential sections through postnatal day 7 cortex revealed low levels of alpha 7 messenger RNA expression delineating the primary sensory areas in layer IV, corresponding to acetylcholinesterase-labeled thalamocortical afferents. However, these sensory areas exhibited higher levels of alpha 7 messenger RNA expression and were more clearly defined in layer VI, but not by acetylcholinesterase staining. The distribution of alpha 7 messenger RNA within the developing thalamocortical system parallels the distribution of alpha-bungarotoxin binding sites and suggests that the receptor is localized on both thalamic cells and their cortical target neurons. This transient and distinct pattern of distribution of the alpha 7 neuronal nicotinic receptor, which coincides with the major phase of thalamocortical development, suggests that it may play a functional role in the development of cortical circuitry.

Long-term changes in striatal opioid systems after 6-hydroxydopamine lesion of rat substantia nigra.

The effects of unilateral 6-hydroxydopamine lesion of the nigrostriatal pathway on striatal opioid peptides and receptors were determined at different time-intervals, from three days up to 24 weeks, post-lesion. Mu, delta and kappa opioid binding site densities in the ipsilateral caudate-putamen were decreased by 25-50% in rats which exhibited a greater than 90% loss of dopamine uptake sites. Differentiation of radioligand binding to kappa1 and kappa2 subtypes demonstrated a selective loss of kappa2 sites post-lesion. The onset of significant 6-hydroxydopamine lesion-induced changes in striatal opioid binding sites was delayed with respect to the loss of dopamine uptake sites. Furthermore, maximal loss of dopamine uptake sites was apparent within seven days post-lesion, but not until two to four weeks for mu, delta and kappa sites. In animals which exhibited an incomplete loss of dopamine uptake sites (less than 80%) there was no significant change in opioid binding site density. Striatal proenkephalin and prodynorphin messenger RNA levels were increased and decreased, respectively, after complete 6-hydroxydopamine lesion. Modulation of peptide messenger RNA levels was apparent within seven days and was maintained up to 24 weeks post-lesion. In contrast, proenkephalin and prodynorphin messenger RNA levels were unchanged in animals which exhibited an incomplete loss of striatal dopamine uptake sites. Taken together, these observations suggest that the majority of mu, delta and kappa2 opioid binding sites are localized on non-dopaminergic elements in the caudate-putamen, but that substantia nigra innervation plays a role in the control of striatal opioid receptor expression. The 6-hydroxydopamine lesion-induced decreases in striatal opioid binding site density may, in part, be a function of agonist-induced receptor downregulation. Alternatively, both opioid receptor and peptide expression in the caudate-putamen may be directly, but independently, regulated by ventral mesencephalic neurons.

Pituitary cell phenotypes involve cell-specific Pit-1 mRNA translation and synergistic interactions with other classes of transcription factors.

Development of the anterior pituitary gland involves proliferation and differentiation of ectodermal cells in Rathke's pouch to generate five distinct cell types that are defined by the trophic hormones they produce. A detailed ontogenetic analysis of specific gene expression has revealed novel aspects of organogenesis in this model system. The expression of transcripts encoding the alpha-subunit common to three pituitary glycoprotein hormones in the single layer of somatic ectoderm on embryonic day 11 established that primordial pituitary cell commitment occurs prior to formation of a definitive Rathke's pouch. Activation of Pit-1 gene expression occurs as an organ-specific event, with Pit-1 transcripts initially detected in anterior pituitary cells on embryonic day 15. Levels of Pit-1 protein closely parallel those of Pit-1 transcripts without a significant lag. Unexpectedly, Pit-1 transcripts remain highly expressed in all five cell types of the mature pituitary gland, but the Pit-1 protein is detected in only three cell types--lactotrophs, somatotrophs, and thyrotrophs and not in gonadotrophs or corticotrophs. The presence of Pit-1 protein in thyrotrophs suggests that combinatorial actions of specific activating and restricting factors act to confine prolactin and growth hormone gene expression to lactotrophs and somatotrophs, respectively. A linkage between the initial appearance of Pit-1 protein and the surprising coactivation of prolactin and growth hormone gene expression is consistent with the model that Pit-1 is responsible for the initial transcriptional activation of both genes. The estrogen receptor, which has been reported to be activated in a stereotypic fashion subsequent to the appearance of Pit-1, appears to be capable, in part, of mediating the progressive increase in prolactin gene expression characteristic of the mature lactotroph phenotype. This is a consequence of synergistic transcriptional effects with Pit-1, on the basis of binding of the estrogen receptor to a response element in the prolactin gene distal enhancer. These data imply that both transcriptional and post-transcriptional regulation of Pit-1 gene expression and combinatorial actions with other classes of transcription factors activated in distinct temporal patterns, are required for the mature physiological patterns of gene expression that define distinct cell types within the anterior pituitary gland.