targeted-toxins

Kar S, Hawkes C, Jhamandas JH (2004) Selective loss of basal forebrain cholinergic neurons by 192 IgG-saporin induces activation of glycogen synthase kinase-3β activity. Neuroscience 2004 Abstracts 92.2. Society for Neuroscience, San Diego, CA.

Summary: Glycogen synthase kinase-3β (GSK-3β) is a multifunctional enzyme involved in a variety of biological events including development, glucose metabolism and cell death. Its activity is negatively regulated by phosphorylation of Ser9 and upregulated by Tyr216 phosphorylation. Activation of GSK-3β induces apoptosis in a variety of cultured neurons and the inhibitory control of its activity by Akt kinase is one of the best characterized cell survival signaling pathways. In the present study, the cholinergic immunotoxin 192-IgG saporin was used to address the potential role of GSK-3β in the degeneration of the basal forebrain cholinergic neurons which are preferentially vulnerable in Alzheimer’s disease (AD) brain. Our results show that GSK-3β colocalizes with a subset of the forebrain cholinergic neurons and that loss of these neurons is accompanied by a transient decrease in phospho-Akt and phospho-Ser9 GSK-3β levels in the basal forebrain, hippocampus and the cortex. Neither total Akt, GSK-3β, nor phospho-Tyr216 GSK-3β levels were significantly altered in the aforesaid brain regions of treated animals. These results provide the very first evidence that increased GSK-3β activity is associated with in vivo degeneration of the forebrain cholinergic neurons and thus may be involved in the loss of these neurons as observed in AD brains.

Related Products: 192-IgG-SAP (Cat. #IT-01)

Kolasa K, Parsons D, Conger K, Harrell LE (2004) Neurotrophic modulation of cholinergic denervation and hippocampal sympathetic ingrowth following immunolesioning with 192 IgG-saporin. Neuroscience 2004 Abstracts 92.9. Society for Neuroscience, San Diego, CA.

Summary: Injection of specific cholinotoxin, 192 IgG-saporin into the medial septum (MS)of rat induces not only a selective cholinergic denervation of hippocampus (CD),but an ingrowth of peripheral sympathetic fibers, originating from the superior cervical ganglion,into the hippocampus (HSI).A similar process,in which sympathetic noradrenergic axons invade hippocampus,may also occur in Alzheimer’s disease(AD). The severity of cognitive decline in AD patients has been linked to multiple factors including cholinergic and neurotrophic factors and their receptors, which undergo selective alterations throughout the progression of AD.It is known that the sites of neurotrophin synthesis in the septo-hippocampal system are predominantly hippocampal neurons. By using 192 IgG-saporin we have been able to mimic some of the cardinal features of AD e.x.cholinergic denervation and hippocampal sympathetic ingrowth and study their effect on growth factors in dorsal hippocampus. Thus,12 weeks after injection of 192 IgG-saporin we measured neurotrophic protein and mRNA expression using Western blot and RT-PCR techniques,respectively. Choline acetyltransferase activity(ChAT)and norepinephrine(NE) concentration was also detected.There was no change in NGF,BDNF,NT3,GDNF mRNA expression,but we have found significant decrease in 240 bp and increase in 328 bp of persephin mRNA expression in CD, and “normalization” in HSI group. No significant alteration was found in NGF and persephin protein expression, but significant decrease in mature form of BDNF protein expression was found in CD, with “normalization”in HSI group.Results of the study suggest that growth factors are affected by cholinergic denervation and may play an important role in regulation and development of HSI,which might be a beneficial phenomenon for restoration of at least some cognitive function.

Related Products: 192-IgG-SAP (Cat. #IT-01)

Milstein JA, Lehmann O, Theobald DEH, Dalley JW, Robbins TW (2004) Role of noradrenergic mechanisms in sustained attention, impulse control, and effects of methylphenidate in rats: Possible relevance to ADHD. Neuroscience 2004 Abstracts 123.6. Society for Neuroscience, San Diego, CA.

Summary: There has been renewed interest in noradrenergic (NA) modulation of sustained attention and impulse control both clinically, with the approval of the SNRI atomoxetine for the treatment of attention deficit hyperactivity disorder, as well as preclinically, in the mediation of the psychomotor effects of stimulants, where blockade of α1 adrenoreceptors counteracts the locomotor stimulant effects of d-amphetamine. The current study examines the role of NA in the modulation of sustained attention and impulse control using the 5-choice serial reaction time task (5CSRT) in rats. Experiment 1 examined the systemic antagonism of methylphenidate (MP)-induced impulsivity with either prazosin, an α1 adrenoreceptor antagonist, which antagonises the locomotor activating effects of amphetamine, or propranolol, a general β-adrenoreceptor blocker. Prazosin partially attenuated the MP-mediated increase in premature responding, but also caused generalised motor slowing, increasing both correct latency as well as latency to collect food reward. Propranolol completely abolished MP-induced impulsivity. This effect was centrally rather than peripherally mediated, as nadolol, a peripheral β-blocker failed to affect MP-induced premature responding. Other experiments examined the comparative effects of selective dopaminergic or serotonergic receptor blockade. A second experiment investigated the effects of selective anti-DBH saporin-induced prefrontal NA depletion. Animals with prefrontal depletions were unimpaired on the baseline version of the 5CSRT. However, they appeared to be slightly impaired under high event rate conditions. Effects of selective prefrontal NA depletion on MP-induced behavioural changes will also be examined. Taken together, these studies provide evidence for a role of noradrenaline in impulse control and the effects of MP.

Related Products: Anti-DBH-SAP (Cat. #IT-03)

Q: Why do your directions for SSP-SAP (Cat. #IT-11) state that it is to be used within hours after dissolution? To my knowledge, both proteins and peptides are stable in clean solution.

A: In fact, the two components of SSP-SAP (Stable Substance P and Saporin) are quite stable. However, we have found that many things happen in laboratories and some of them can impact stability. Probably the most severe is the loss of sterility. In that case, over time at room temperature or at 4°C, bacteria can grow on this rather excellent “medium.” This would cause inactivation. Because many laboratories, due to molecular biology work, have high levels of resident bacteria, we prefer to emphasize playing it safe.

Even if saporin is a stable protein, it is a protein and can suffer denaturation. This occurs more rapidly at room temperature than at 4°C, and hardly at all in the frozen state (really, it is stable for years when stored at -80°C). The maintenance of precise activity is of extreme importance to our customers who use these materials in vivo (their assays are very sensitive), and so we choose to advise the most conservative course.

Q: I understand that theoretically only one molecule of Saporin taken up by a cell is enough to induce cell death. I have been looking for literature on this topic but have not come across anything.

A: Definitely theoretical. The only article that we know of that states anything close to that is: Yamaizumi et al (1978) One molecule of diphtheria toxin fragment A introduced into a cell can kill the cell. Cell 15(1): 245-250. As you can see, this article speaks to the enzymatic chain of diphtheria toxin, which has a slightly different mechanism of action for shutting down protein synthesis, but otherwise is similar to saporin. In fact, we test all sorts of toxins against cells in controlled conditions, and we have only one candidate that is in this range; all the rest are orders of magnitude away. It takes more than thousands per cell. Another question would be: how many actually get in?

In another FAQ, you addressed the question of one molecule of saporin killing a cell. Your response overlooked the data on ricin, abrin and modeccin (Eiklid, Olsnes and Pihl, Exp Cell Res, 126:321-326, 1980). In that paper, they showed that these RIP toxins applied to cells in culture produce all-or-none lethality. They used radioactive amino acid uptake and incorporation (as memory serves) and found only two types of cells, those with absolutely no uptake of label or those that were entirely normal – nothing in between. Also, if the data on ricin-induced apoptosis is correct (numerous authors), and I believe it is, then at low doses, the cells die from triggering apoptosis which seem possible with a single molecule of RIP free in the cytoplasm. To further complete your answer, someone (I haven’t found the article yet) showed that it took, on average, about 10,000 molecules of ricin/cell to kill cells in culture. This gives a hint at the efficiency of internalization and translocation in that cell type. I am not aware anyone else has looked at these issues with saporin conjugates.

A: Overlooking literature is actually a favorite sport of mine, but in this case I would respectfully point out conflicting information. There is a study of something that is quite between an all-or-none phenomenon: Barbieri et al. FEBS Lett, 2003 Mar 13;538(1-3):178-82. These authors document that ribosome-inactivating proteins have transforming activity on the classic FDA assay cell line: NIH3T3 cells. This would be a non-toxic activity that one presumes is due to internalization, and is somewhat on the none side of all or none, but hey, it’s an activity nonetheless. 

See: Targeted Toxins

Yoshimura N (2004) Featured Article: Effects of IB4-SAP on bladder overactivity. Targeting Trends 5(4)

Related Products: IB4-SAP (Cat. #IT-10)

Read the featured article in Targeting Trends.

See Also:

• Nishiguchi J et al. Effects of isolectin B4-conjugated saporin, a targeting cytotoxin, on bladder overactivity induced by bladder irritation. Eur J Neurosci 20(2):474-482, 2004.

Cameron NM, Carey P, Erskine MS (2004) Medullary noradrenergic neurons release norepinephrine in the medial amygdala in females in response to mating stimulation sufficient for pseudopregnancy. Brain Res 1022(1-2):137-147. doi: 10.1016/j.brainres.2004.07.022

Summary: Norepinephrine (NE) plays an important role in female reproductive function. While the ventral noradrenergic bundle is known to be necessary for transmitting the pseudopregnancy (PSP) response, the mechanism by which this occurs is not understood. The authors administered 20 ng of Anti-DBH-SAP (Cat. #IT-03) to the left posterodorsal medial amygdala of ovariectomized rats. The results indicate that NE may play an important role in the establishment of PSP.

Related Products: Anti-DBH-SAP (Cat. #IT-03)

Gerashchenko D, Chou TC, Blanco-Centurion CA, Saper CB, Shiromani PJ (2004) Effects of lesions of the histaminergic tuberomammillary nucleus on spontaneous sleep in rats. Sleep 27(7):1275-1281. doi: 10.1093/sleep/27.7.1275

Summary: Although evidence suggests that histaminergic neurons in the tuberomammillary nucleus (TMN) promote wakefulness, this has not been investigated using specific lesioning agents. In this study, the authors utilize the fact that TMN neurons express the orexin-B receptor by eliminating these neurons with an injection of 50 ng of orexin-SAP (Cat. #IT-20) into the posterior hypothalamus. The data indicate that histaminergic neurons are not required for the homeostatic regulation of sleep.

Related Products: Orexin-B-SAP (Cat. #IT-20)

Hudson B, Ritter S (2004) Hindbrain catecholamine neurons mediate consummatory responses to glucoprivation. Physiol Behav 82(2-3):241-250. doi: 10.1016/j.physbeh.2004.03.032

Summary: Norepinephrine (NE) and epinephrine (E) neurons appear to potently stimulate feeding behavior when administered to the hypothalamus. Previous work has indicated that these neurons play important roles in feeding responses due to glucoprivation. Bilateral 42 ng-injections of anti-DBH-SAP (Cat. #IT-03) were administered to rats to investigate the roles of NE and E neurons in the consummatory phase of the glucoprivic response. The results indicate that catecholaminergic neurons are involved in both appetitive and consummatory responses to glucoprivation.

Related Products: Anti-DBH-SAP (Cat. #IT-03)

Ricceri L, Minghetti L, Moles A, Popoli P, Confaloni A, De Simone R, Piscopo P, Scattoni ML, di Luca M, Calamandrei G (2004) Cognitive and neurological deficits induced by early and prolonged basal forebrain cholinergic hypofunction in rats. Exp Neurol 189(1):162-172. doi: 10.1016/j.expneurol.2004.05.025

Summary: A distinctive feature of Alzheimer’s disease is the loss of cholinergic neurons in the basal forebrain (BF). The authors investigated long-term effects of BF cholinergic lesions on several parameters. Administration of 0.21 µg of 192-Saporin (Cat. #IT-01) to the third ventricle of 7 day-old rats was followed by an evaluation of protein levels and cortical EEG patterns at 6 months of age. The findings indicate that permanent neonatal BF cholinergic damage may provide a model for abnormal adult cholinergic function.

Related Products: 192-IgG-SAP (Cat. #IT-01)

Kudoh M, Seki K, Shibuki K (2004) Sound sequence discrimination learning is dependent on cholinergic inputs to the rat auditory cortex. Neurosci Res 50(1):113-123. doi: 10.1016/j.neures.2004.06.007

Summary: The auditory cortex (AC) is thought to play a role in the discrimination of sound sequences. The authors investigated the role of cholinergic inputs to the AC in processing these sequences by injecting 5 µg of 192-Saporin (Cat. #IT-01) into either the lateral ventricle or bilateral AC of rats. Treated animals displayed suppressed sound discrimination learning, but discrimination between two sound components was unaffected. The results suggest that cholinergic neurons in the AC are highly involved in sound sequence learning.

Related Products: 192-IgG-SAP (Cat. #IT-01)