Destruction of midbrain dopaminergic neurons by using immunotoxin to dopamine transporter.

1. The ability to target specific neurons can be used to produce selective neural lesions and potentially to deliver therapeutically useful moieties for treatment of disease. In the present study, we sought to determine if a monoclonal antibody to the dopamine transporter (anti-DAT) could be used to target midbrain dopaminergic neurons. 2. The monoclonal antibody recognizes the second, large extracellular loop of DAT. The antibody was conjugated to the "ribosome-inactivating protein"; saporin, and stereotactically pressure microinjected into either the center of the striatum or the left lateral ventricle of adult, male Sprague-Dawley rats. 3. Local intrastriatal injections produced destruction of dopaminergic neurons in the ipsilateral substantia nigra consistent with suicide transport of the immunotoxin. Intraventricular injections (i.c.v.) produced significant loss of dopaminergic neurons in the substantia nigra and ventral tegmental area bilaterally without evident damage to any other aminergic structures such as the locus coeruleus and raphe nuclei. To confirm the anatomic findings, binding of [3-H]mazindol to DAT in the striatum and midbrain was assessed using densitometric analysis of autoradiograms. Anti-DAT-saporin injected i.c.v. at a dose of 21 microg, but not 8 microg, produced highly significant decreases in mazindol binding consistent with loss of the dopaminergic neurons. 4. These results show that anti-DAT can be used to target midbrain dopaminergic neurons and that anti-DAT-saporin may be useful for producing a lesion very similar to the naturally occurring neural degeneration seen in Parkinson's disease. Anti-DAT-saporin joins the growing list of neural lesioning agents based on targeted cytotoxins.

Selective immunolesions of cholinergic neurons in mice: effects on neuroanatomy, neurochemistry, and behavior.

The ability to selectively lesion mouse basal forebrain cholinergic neurons would permit experimental examination of interactions between cholinergic functional loss and genetic factors associated with neurodegenerative disease. We developed a selective toxin for mouse basal forebrain cholinergic neurons by conjugating saporin (SAP), a ribosome-inactivating protein, to a rat monoclonal antibody against the mouse p75 nerve growth factor (NGF) receptor (anti-murine-p75). The toxin proved effective and selective in vitro and in vivo. Intracerebroventricular injections of anti-murine-p75-SAP produced a dose-dependent loss of choline acetyltransferase (ChAT) activity in the hippocampus and neocortex without affecting glutamic acid decarboxylase (GAD) activity. Hippocampal ChAT depletions induced by the immunotoxin were consistently greater than neocortical depletions. Immunohistochemical analysis revealed a dose-dependent loss of cholinergic neurons in the medial septum (MS) but no marked loss of cholinergic neurons in the nucleus basalis magnocellularis after intracerebroventricular injection of the toxin. No loss of noncholinergic neurons in the MS was apparent, nor could we detect loss of noncholinergic cerebellar Purkinje cells, which also express p75. Behavioral analysis suggested a spatial learning deficit in anti-murine-p75-SAP-lesioned mice, based on a correlation between a loss of hippocampal ChAT activity and impairment in Morris water maze performance. Our results indicate that we have developed a specific cholinergic immunotoxin for mice. They also suggest possible functional differences in the mouse and rat cholinergic systems, which may be of particular significance in attempts to develop animal models of human diseases, such as Alzheimer's disease, which are associated with impaired cholinergic function.

Hypocretin-2-saporin lesions of the lateral hypothalamus produce narcoleptic-like sleep behavior in the rat.

Hypocretins (Hcrts) are recently discovered peptides linked to the human sleep disorder narcolepsy. Humans with narcolepsy have decreased numbers of Hcrt neurons and Hcrt-null mice also have narcoleptic symptoms. Hcrt neurons are located only in the lateral hypothalamus (LH) but neither electrolytic nor pharmacological lesions of this or any other brain region have produced narcoleptic-like sleep, suggesting that specific neurons need to be destroyed. Hcrt neurons express the Hcrt receptor, and to facilitate lesioning these neurons, the endogenous ligand hypocretin-2/orexin B (Hcrt2) was conjugated to the ribosome-inactivating protein saporin (SAP). In vitro binding studies indicated specificity of the Hcrt2-SAP because it preferentially bound to Chinese hamster ovary cells containing the Hcrt/orexin receptor 2 (HcrtR2/OX(2)R) or the Hcrt/orexin receptor 1 (HcrtR1/OX(1)R) but not to Kirsten murine sarcoma virus transformed rat kidney epithelial (KNRK) cells stably transfected with the substance P (neurokinin-1) receptor. Administration of the toxin to the LH, in which the receptor is known to be present, eliminated some neurons (Hcrt, melanin-concentrating hormone, and adenosine deaminase-containing neurons) but not others (a-melanocyte-stimulating hormone), indicating specificity of the toxin in vivo. When the toxin was administered to the LH, rats had increased slow-wave sleep, rapid-eye movement (REM) sleep, and sleep-onset REM sleep periods. These behavioral changes were negatively correlated with the loss of Hcrt-containing neurons but not with the loss of adenosine deaminase-immunoreactive neurons. These findings indicate that damage to the LH that also causes a substantial loss of Hcrt neurons is likely to produce the multiple sleep disturbances that occur in narcolepsy.

Inhibition of neuropathic pain by selective ablation of brainstem medullary cells expressing the mu-opioid receptor.

Neurons in the rostroventromedial medulla (RVM) project to spinal loci where the neurons inhibit or facilitate pain transmission. Abnormal activity of facilitatory processes may thus represent a mechanism of chronic pain. This possibility and the phenotype of RVM cells that might underlie experimental neuropathic pain were investigated. Cells expressing mu-opioid receptors were targeted with a single microinjection of saporin conjugated to the mu-opioid agonist dermorphin; unconjugated saporin and dermorphin were used as controls. RVM dermorphin-saporin, but not dermorphin or saporin, significantly decreased cells expressing mu-opioid receptor transcript. RVM dermorphin, saporin, or dermorphin-saporin did not change baseline hindpaw sensitivity to non-noxious or noxious stimuli. Spinal nerve ligation (SNL) injury in rats pretreated with RVM dermorphin-saporin failed to elicit the expected increase in sensitivity to non-noxious mechanical or noxious thermal stimuli applied to the paw. RVM dermorphin or saporin did not alter SNL-induced experimental pain, and no pretreatment affected the responses of sham-operated groups. This protective effect of dermorphin-saporin against SNL-induced pain was blocked by beta-funaltrexamine, a selective mu-opioid receptor antagonist, indicating specific interaction of dermorphin-saporin with the mu-opioid receptor. RVM microinjection of dermorphin-saporin, but not of dermorphin or saporin, in animals previously undergoing SNL showed a time-related reversal of the SNL-induced experimental pain to preinjury baseline levels. Thus, loss of RVM mu receptor-expressing cells both prevents and reverses experimental neuropathic pain. The data support the hypothesis that inappropriate tonic-descending facilitation may underlie some chronic pain states and offer new possibilities for the design of therapeutic strategies.

Targeted toxins.

Molecular neurosurgery can be used to make selective neural lesions by targeting cytotoxins to specific populations of neurons based on their common expression of a particular surface molecule. The targeted toxins employed in this unit consist of a targeting moiety (vector) and an effector moiety (cytotoxin). In all cases discussed in this unit, the cytotoxic moiety is an enzyme that catalytically inactivates the large ribosomal subunit, irreversibly inhibiting protein synthesis and resulting in cell death. These toxins appear to kill in an all-or-none fashion, indicating that one molecule of free cytotoxin in the cytoplasm of a cell is sufficient to kill the cell. Three general molecular neurosurgery protocols are presented in this unit. The first describes suicide transport, which refers to the use of targeted toxins to make anatomically restricted lesions based on retrograde axonal transport of the toxin. The second involves immunolesioning and uses anti-neuronal immunotoxins to make type-selective and anatomically restricted lesions. The final protocol uses neuropeptide-toxin conjugates to selectively destroy neurons expressing the receptor for the specific neuropeptide.

Entering through the doors of perception: characterization of a highly selective Substance P receptor-targeted toxin.

Perception of external stimuli is often mediated through the activity of a G protein-coupled receptor in response to its ligand. Receptor-mediated internalization allows the insertion of toxins that cause the elimination of receptor-expressing neurons. Using this technique new information on systems biology can be discovered and with this, new therapeutics developed.

Schwann cells are removed from the spinal cord after effecting recovery from paraplegia.

Remyelination of the CNS is necessary to restore neural function in a number of demyelinating conditions. Schwann cells, the myelinating cells of the periphery, are candidates for this purpose because they have more robust regenerative properties than their central homologs, the oligodendrocytes. Although the ability of Schwann cells to remyelinate the CNS has been demonstrated, their capacity to enter the adult spinal cord in large numbers and effect functional recovery remains uncertain. We used cholera toxin B-subunit conjugated to saporin to demyelinate the rat lumbar spinal cord, remove macroglia, and produce paraplegia. After the removal of oligodendrocyte and astrocyte debris by invading macrophages, there was a spontaneous entry of Schwann cells into the spinal cord, along with axonal remyelination and concomitant functional recovery from paraplegia occurring within 75 d. The Schwann cells appeared to enter the dorsal funiculi via the dorsal root entry zone and the lateral funiculi via rootlets that had become adherent to the lateral spinal cord after the inflammation. In the following weeks, Schwann cell myelin surrounding central axons was progressively replaced by oligodendrocyte myelin without lapse in motor function. Our results show that endogenous Schwann cells can reverse a severe neurological deficit caused by CNS demyelination and enable later oligodendrocyte remyelination.

Mab-ZAP: a tool for evaluating antibody efficacy for use in an immunotoxin.

Immunotoxins, consisting of antibodies coupled to toxins, are extremely useful tools in the elimination of specific cell populations in vitro and in vivo for research and therapeutic applications. The antibody is used to target the toxin to a specific cell population, which is distinguished by its cell-surface antigen. Not all antibodies are suitable for creating an immunotoxin, and large numbers of antibodies may need to be screened. This is a time-consuming and expensive process if each potential candidate must be conjugated to the toxin and purified. A faster and more economical way to identify potential targeting antibodies is to use a second immunotoxin, an anti-IgG antibody that is coupled to the toxin. The second immunotoxin eliminates the need to couple every candidate antibody to the toxin because it can simply be added to cells in culture with the antibody of interest. Using this method, many antibodies can be screened quickly and efficiently for their ability to internalize.

Selective impairment of corticotropin-releasing factor1 (CRF1) receptor-mediated function using CRF coupled to saporin.

CRF is the main component in the brain neuropeptide effector system responsible for the behavioral, endocrine, and physiological activation that accompanies stress activation. Reduced CRF system activation plays a role in the etiology of a variety of psychiatric and metabolic disease states. We have developed a novel protein conjugate that joins native rat/human CRF to a ribosome-inactivating protein, saporin (CRF-SAP), for the purpose of targeted inactivation of CRF receptor-expressing cells. Cytotoxicity measurements revealed that CRF-SAP (1-100 nM) produced concentration-dependent and progressive cell death over time in CRF1 receptor-transfected L cells, but at similar concentrations had no effect on CRF2alpha receptor-transfected cells. The CRF-SAP-induced toxicity in CRF1-transfected cells was prevented by coincubation with the competitive CRF1/CRF2 receptor peptide antagonist, [D-Phe12]CRF-(12-41), or the selective nonpeptide CRF1 receptor antagonist, NBI 27914. Finally, in cultured rat pituitary cells that express native CRF1 receptors, CRF-SAP suppressed CRF-induced (1 nM) ACTH release. GnRH (1-10 nM) stimulated LH release was also assessed in the same pituitary cultures. Although there was a slight decrease in LH release from these cultures, this decrease was observed with CRF-SAP or SAP alone, suggesting that the response was nonspecific. Taken together, these results suggest the utility of CRF-SAP as a specific and subtype-selective tool for long term impairment of CRF1 receptor-expressing cells.

Transmission of chronic nociception by spinal neurons expressing the substance P receptor.

Substance P receptor (SPR)-expressing spinal neurons were ablated with the selective cytotoxin substance P-saporin. Loss of these neurons resulted in a reduction of thermal hyperalgesia and mechanical allodynia associated with persistent neuropathic and inflammatory pain states. This loss appeared to be permanent. Responses to mildly painful stimuli and morphine analgesia were unaffected by this treatment. These results identify a target for treating persistent pain and suggest that the small population of SPR-expressing neurons in the dorsal horn of the spinal cord plays a pivotal role in the generation and maintenance of chronic neuropathic and inflammatory pain.

Threshold relationship between lesion extent of the cholinergic basal forebrain in the rat and working memory impairment in the radial maze.

The cholinergic basal forebrain (CBF) degenerates in Alzheimer's Disease (AD), and the degree of this degeneration correlates with the degree of dementia. In the present study we have modeled this degeneration in the rat by injecting various doses of the highly selective immunotoxin 192 IgG-saporin (192-sap) into the ventricular system. The ability of 192-sap-treated rats to perform in a previously learned radial maze working memory task was then tested. We report here that 192-sap created lesions of the CBF and, to a lesser extent, cerebellar Purkinje cells in a dose-dependent fashion. Furthermore, we found that rats harboring lesions of the entire CBF greater than 75% had impaired spatial working memory in the radial maze. Correlational analysis of working memory impairment and lesion extent of the component parts of the CBF revealed that high-grade lesions of the hippocampal-projecting neurons of the CBF were not sufficient to impair working memory. Only rats with high-grade lesions of the hippocampal and cortical projecting neurons of the CBF had impaired working memory. These data are consistent with other 192-sap reports that found behavioral deficits only with high-grade CBF lesions and indicate that the relationship between CBF lesion extent and working memory impairment is a threshold relationship in which a high degree of neuronal loss can be tolerated without detectable consequences. Additionally, the data suggest that the CBF modulates spatial working memory via its connections to both the hippocampus and cortex.

Targeting neurokinin-1 receptor-expressing neurons with [Sar9,Met(O2)11 substance P-saporin.

Neurons expressing neurokinin-1 receptors (NK-1R) are selectively destroyed by substance P (SP) coupled to the ribosome inactivating protein, saporin. SP-saporin produces incomplete lesions of striatal NK-1R-expressing neurons even at doses that produce non-specific damage. In the present study, we sought to determine if the more stable, NK-1R-specific SP analog conjugated to saporin, [Sar9,Met(O2)11]-SP (SSP-saporin), would selectively destroy cells expressing NK-1R, in vitro and in vivo. The results show that SSP-saporin is highly effective and selective, producing extensive ablation of striatal NK-1R expressing interneurons at doses that do not cause loss of other striatal neurons suggesting advantages over SP-saporin as a selective lesioning agent. SSP-saporin will be useful in larger species and for intraparenchymal injections.

Selective lesion of the cholinergic basal forebrain causes a loss of cortical neuropeptide Y and somatostatin neurons.

Degeneration of the cholinergic basal forebrain (CBF) and changes in cortical neuropeptide levels have been reported in Alzheimer's disease. In the present study, we sought to determine if a selective cholinergic lesion of nucleus basalis magnocellularis (Nbm) could affect the number and distribution of neuropeptide Y (NPY) and somatostatin (SS) immunoreactive neurons in the frontoparietal and occipital cortices of rats. Brain sections were evaluated at survival times of 1, 2, 4, 8, 12, 24, 48, 78 and 100 weeks after intraventricular injection of 192-saporin, an immunotoxin directed at the low affinity neurotrophin receptor (p75NGFr), that selectively destroys the CBF. Following the immunotoxin lesion of the Nbm, the number of NPY-labeled neurons decreased 33% in the frontoparietal cortex and 60% in the occipital cortex compared to age-matched normal controls at most survival time points. A significant loss of SS-labeled neurons in both cortical regions was seen 12 weeks after 192-saporin injection with no further change up to 100-week survival time. The effect of age on neuropeptidergic populations was evaluated in normal control rats. The number of NPY and SS immunoreactive neurons in aged rats (21-26 months) decreased by 42% in the frontoparietal cortex and 27% in the occipital cortex when compared with young (3-6 months) and middle-age (9-14 months) rats. When both non-lesioned and lesioned animals with different ages were pooled for linear regression, a significant correlation was found between the number of cortical NPY- and SS-labeled neurons and cortical acetylcholinesterase (AChE) histochemical staining intensity. These findings indicate that: (1) cholinergic denervation of the Nbm is associated with an irreversible loss of neocortical NPY and SS immunoreactive neurons analogous to that observed in Alzheimer's disease and aging; (2) the degree of the loss of cortical NPY and SS immunoreactive neurons seems to be related to the extent of the reduction of cortical AChE intensity in both toxin-injected and normal aged rats. These findings may reflect a trophic dependence of NPY and SS neurons on cortical cholinergic input.

Destruction of locus coeruleus neuronal perikarya after injection of anti-dopamine-B-hydroxylase immunotoxin into the olfactory bulb of the rat.

Saporin, a ribosome-inactivating protein, was coupled to a monoclonal antibody to dopamine-B-hydroxylase (DBH) and injected unilaterally into the olfactory bulb of rats. After 4-13 days survival, the rat brain was processed histologically and the locus coerulei (LC) examined with Nissl and anti-DBH staining. There were degenerating dendrites in surviving LC neurons on the side ipsilateral to the immunotoxin-injected olfactory bulb. The number of Nissl-positive LC neurons in a transverse section through the caudal one third of the LC was reduced from 116+/-10 to 50+/-8 neurons (P < 0.01, n = 7) and the number of DBH-positive neurons in the more rostral LC sections was reduced from 13+/-2 to 5+/-1 (P < 0.05, n = 4). Our results indicate that it is possible to lesion LC neurons via retrograde intraaxonal transport of saporin-anti-DBH immunotoxin from the olfactory bulb.

Inhibition of hyperalgesia by ablation of lamina I spinal neurons expressing the substance P receptor.

Substance P is released in the spinal cord in response to painful stimuli, but its role in nociceptive signaling remains unclear. When a conjugate of substance P and the ribosome-inactivating protein saporin was infused into the spinal cord, it was internalized and cytotoxic to lamina I spinal cord neurons that express the substance P receptor. This treatment left responses to mild noxious stimuli unchanged, but markedly attenuated responses to highly noxious stimuli and mechanical and thermal hyperalgesia. Thus, lamina I spinal cord neurons that express the substance P receptor play a pivotal role in the transmission of highly noxious stimuli and the maintenance of hyperalgesia.

Destruction of neurokinin-1 receptor expressing cells in vitro and in vivo using substance P-saporin in rats.

Substance P (SP) acts on neurons through the neurokinin-1 (NK-1) receptor. Conjugation of SP to the ribosome inactivating protein, saporin (SAP), produces a cytotoxin selective for cells that express the NK-1 receptor. SP-SAP cytotoxicity was inhibited by pre-treating the toxin to reduce the disulfide bond connecting SP to SAP or by pre-incubation with anti-SP antiserum or by SP analog showing that SP-SAP acts through binding of the SP moiety to NK-1 receptors. Injection of SP-SAP into the striatum selectively destroyed NK-1 receptor expressing interneurons. These results show that SP-SAP will be useful for studying the function of NK-1 receptor expressing neurons.

Retrograde degeneration and colchicine protection of basal forebrain cholinergic neurons following hippocampal injections of an immunotoxin against the P75 nerve growth factor receptor.

Intracerebroventricular injection of 192 IgG antibody against the p75LNGFR rat low affinity nerve growth factor receptor conjugated with saporin, a ribosome inactivating protein, has been shown to destroy the p75LNGFR-expressing cholinergic neurons of the basal forebrain. We injected this immunotoxin into the hippocampus and studied its retrograde effect upon the cholinergic neurons of the medial septum and the vertical limb of the diagonal band of Broca. Seven days after injection, there was a nearly total depletion of cholinergic axons within the hippocampus. This depletion was associated with a marked and significant decrease in the number of cholinergic neurons of the ipsilateral medial septum and the vertical limb of the diagonal band of Broca. At longer survival times, these changes were more pronounced. Parvalbumin-positive, GABAergic neurons within the same areas of the basal forebrain were not affected by immunotoxin injections. Injections of saporin alone had no effect upon cholinergic neurons. Simultaneous injection of colchicine with the immunotoxin resulted in a significant reduction of retrograde degeneration of cholinergic neurons and relative preservation of hippocampal cholinergic axons. These observations suggest that 192 IgG-saporin is transported retrogradely from the hippocampus to the cholinergic neurons in the medial septum and the vertical limb of the diagonal band of Broca and provide a model for retrograde degeneration of basal forebrain cholinergic neurons following cortically based toxic-pathologic processes.

Effect of basic fibroblast growth factor-saporin mitotoxin on breast cancer cell lines in vitro.

Fibroblast growth factor receptors are widely expressed on breast cancer cells and we report a preliminary study to determine whether these could be useful as potential targets for delivery of a cytotoxic fibroblast growth factor 2-saporin conjugate. We show that this mitotoxin conjugate can displace I-125-fibroblast growth factor 2 binding though with reduced affinity compared to unlabeled fibroblast growth factor 2. For 4 out of 5 cell lines it is an effective inhibitor of cell growth, and cytotoxic for at least 2 of the lines. Inhibitory effects did not depend on responsiveness of cells to fibroblast growth factor 2. This activity was not achieved with free saporin. There may be potential uses for this conjugate in both experimental systems to study receptor function and subsequent processing, and also in clinical settings to eliminate breast cancer cells.

Central noradrenergic lesioning using anti-DBH-saporin: anatomical findings.

The ability to create lesions of discrete neuronal populations is an important strategy for clarifying the function of these populations. The power of this approach is critically dependent upon the selectivity of the experimental lesioning technique. Anti-neuronal immunotoxins offer an efficient way to produce highly specific neural lesions. Two previous immunotoxins have been shown to be effective in both the CNS and PNS. They are OX7-saporin, which is targeted at Thy1, and 192-saporin, which is targeted at the low affinity neurotrophin receptor, p75NTR. In the present study, we sought to determine if an immunotoxin targeted at the neurotransmitter synthesizing enzyme, dopamine beta-hydroxylase (DBH), could selectively destroy central noradrenergic neurons after intraventricular administration. This immunotoxin, which consists of a monoclonal antibody to DBH coupled by a disulfide bond to saporin (a ribosome inactivating protein), has been shown to be selectively toxic to peripheral noradrenergic sympathetic neurons in rats after systemic injection. In the present study, immunohistochemical and Cresyl violet staining showed that the noradrenergic neurons of the locus coeruleus are destroyed bilaterally after intraventricular (i.c.v.) injection of 5, 10, and 20 micrograms of anti-DBH-saporin (alpha-DBH-sap) into rats. Complete bilateral lesioning of the A5 and A7 cell groups occurred at the two higher doses. Lesions of the A1/C1 and A2/C2/C3 cell groups were incomplete at all three doses. Dopaminergic neurons of the substantia nigra and ventral tegmental area and serotonergic neurons of the raphé, all monoaminergic neurons that do not express DBH, survived all alpha-DBH-sap doses. The cholinergic neurons of the basal forebrain, which are selectively killed by i.c.v. injection of 192-saporin, and cerebellar Purkinje cells which are killed by OX7-saporin, were not killed by alpha-DBH-sap. These results show that alpha-DBH-sap efficiently and selectively destroys CNS noradrenergic neurons after i.c.v. injection. The preferential destruction of locus coeruleus, A5, and A7 over A1/C1 and A2/C2/C3 may be due to more efficient access of the immunotoxin to these neurons and their terminals after i.c.v. injection.

Stimulation of endothelial cells by doses of basic FGF-saporin that are lethal to smooth muscle cells.

Basic fibroblast growth factor (FGF) receptors are up-regulated in proliferating (vs. quiescent) aortic smooth muscle cells, according to the results of recent studies. This up-regulation allows the ribosome inactivator saporin (if linked to basic FGF) to enter and kill proliferating, but not quiescent smooth muscle cells in vitro and in vivo. The authors now report that endothelial cells exhibit a different response. In 10% serum, FGF-SAP (0.1-1 nM) stimulates protein synthesis and cell division in subconfluent endothelial cells, but inhibits protein synthesis and cell division in subconfluent smooth muscle cells. Endothelial cells were inhibited at 10 nM FGF-SAP. A stimulatory response was seen in smooth muscle cells only at 0.1 nM FGF-SAP, and only after serum deprivation. Both cell types were resistant to FGF-SAP at high cell density. These responses correlated with FGF receptor density, which was sixfold higher in smooth muscle than endothelial cells and twice as high in serum-free smooth muscle cells as in serum-deprived smooth muscle cells. Moreover, a dose of FGFSAP that inhibited neointimal smooth muscle accumulation after balloon injury did not inhibit reendothelialization. Thus, there is a dose range at which FGF-SAP has unique properties that may make it useful in the treatment of vascular injury.