Today we’re highlighting one of our saporin conjugates, Kisspeptin-SAP. This is a tool for eliminating cells that express the human and sheep G-protein coupled receptor, GPR54.
Kisspeptin is a ligand to GPR54 and functions in the hypothalamus, placenta, liver, pancreas, adipose tissue, bone, and limbic regions which shows the importance it can play in diagnosis and treatment of various disorders. It is one of the neuropeptides that governs the reproductive endocrine axis by regulating neuronal activity and secretion of hypothalamic gonadotropin releasing hormone (GnRH). GnRH is released from the hypothalamus to act on the anterior pituitary triggering the release of luteinizing hormone (LH), and follicle stimulating hormone (FSH). These gonadotropic hormones lead to sexual maturation and gametogenesis.
This is a 2023 publication using a Kisspeptin-saporin conjugate.
The objective of the authors was to test the functional role of ovine KNDy neurons in pulse generation and identify the roles of nearby Kiss1 receptor (Kiss1R)-containing cells. They performed these tests using conjugates NKB-SAP and Kisspeptin-SAP.
The results showed that NKB-SAP ablated over 90% of the KNDy cells, while Kisspeptin-SAP lesioned about two-thirds of the Kiss1R population. This led to a significant decrease in LH pulse amplitude and altering LH pulse patterns. NK3-SAP increased the interpulse interval without affecting the regularity of LH pulses, whereas Kiss-SAP disrupted their regular hourly occurrence but not the interpulse interval.
These findings suggest that KNDy neurons are critical for GnRH pulse generation in ewes, while ARC Kiss1R cells support the amplitude and regularity of these pulses, possibly as part of a positive feedback loop involving GABA or glutamate.
Today we’re highlighting one of our saporin conjugates, Anti-CD117-SAP. This is a tool for depleting cells that express CD117, the tyrosine kinase growth factor receptor.
CD117 is expressed on hematopoietic stem cells, mast cells, and acute myeloid leukemia cells. Some of the areas where CD117 plays an essential role is in the regulation of cell survival and proliferation, hematopoiesis, stem cell maintenance and mast cell development, migration and function.
Allogeneic hematopoietic stem cell transplantation can cure genetic diseases such as severe combined immunodeficiency (SCID), but it’s associated with significant toxicities such as graft-versus-host disease (GvHD). Using a patients’ own gene-modified hematopoietic stem and progenitor cells (HSPCs) can eliminate the risk of GvHD, but the treatment relies on gene transfer using viruses and genotoxic conditioning which carries risks.
The authors describe how base-editing is an alternative means to correct genetic defects while engineered virus-like particles (eVLPs) can deliver the base-edited proteins without the risks seen with viral integration.
In conclusion, transplantation of HSPCs that have been repaired through eVLP-mediated base-editing with CD117-ADC conditioning successfully reversed the SCID phenotype in mice, and highlights a significant advancement in disease treatment without the current toxicities.
We are highlighting one of our saporin conjugates, Neurotensin-SAP — a tool for depleting cells that express the neurotensin receptor (NTSR).
Neurotensin is a 13-amino-acid peptide found in the brain and spinal cord and is released by the hypothalamus. It is synthesized as part of a larger precursor protein that also includes the related neuropeptide neuromedin N. Neurotensin affects pituitary hormone release, interacts with the dopaminergic system, and is involved in vasodilation and hypotension. It can also modulate pain perception where intrathecal neurotensin has also been shown to be anti-nociceptive.
The objective was to determine whether NTSR1-expressing enteropancreatic neurons mediate the glucose-lowering effects of dietary olive oil and neurotensin, and to characterize their physiological role in glucose homeostasis.
The authors unilaterally injected neurotensin-SAP into the nodose ganglia to ablate NTSR1-expressing vagal neurons.
In their study, they demonstrated that neurotensin improves glucose tolerance by activating NTSR1-expressing enteropancreatic neurons, which connect the gut and pancreas. Ablation or disruption of these neurons abolished the glucoregulatory effects of both neurotensin and olive oil, establishing their necessity and sufficiency in this pathway.
Wang W, Zhang Y, Li L, Xu Y, Zhang W, Chen X, Wang X, Tong G, Zhang P (2026) PACAP/PAC 1 modulates light-induced sleep via the ipRGC-VLPO pathway. Biochem Biophys Res Commun 808:153462. doi: 10.1016/j.bbrc.2026.153462 PMID: 41702189
Objective: To investigate the mechanism of PACAP in ipRGC–VLPO light-induced sleep.
Summary: Partial ablation of ipRGCs by Melanopsin-SAP reduced light-induced sleep duration, whereas PACAP 1-38 administration reversed this effect, leading to an increase in REM sleep. After the partial destruction of ipRGCs through the intraocular injection of saporin (SAP), we continued to observe the effect of PACAP on light-induced sleep. The results showed that after the microdialysis injection of PACAP 1-38 into the VLPO of SAP mice, the light-induced sleep of the mice increased; specifically, REM sleep significantly increased. The results suggest that PACAP is involved in ipRGC–VLPO-mediated light-induced sleep.
Today’s topic is time-course for our saporin conjugates. Common questions we get are (1) how long does it takes to see cell death, (2) how long should I wait before performing histology on an animal, or (3) how long before I see behavioral changes in an animal?
In this image from Mantyh et. al. (1997), we are looking at confocal imagery of the binding and internalization of our peptide conjugate SP-SAP to the NK1r receptor in primary cultures of neonatal spinal cord neurons.
As you can see, conjugate binding occurs immediately where within hours, the SP-SAP has recognized and bound to the NK1 receptor. Here we see the areas of concentrated NK1R expression marked by yellow immunofluorescence.
But how long until you see cell death? Here is a cytotoxicity graph of in vitro data of our antibody conjugate 192-IgG-SAP. These are typical data after cells have been treated for 3 days, which is standard protocol.
Waite et al. (1994) used 192-IgG-SAP and showed the appearance of behavioral effects associated with neuronal loss at day four and plateauing at day 7.
This coincides with the time course seen in vitro. At this point, microglia will infiltrate, but stop at 7 days, which is probably the peak day for infiltration. Once there is complete removal of the detritus, microglia down-regulate and at 14 days all debris is cleared and the animal begins to regain normal eating and sleeping habits. This is the idea behind waiting about 2 weeks before performing histology.
Doyle S, Doran MM, Cunningham C, Lowry JP (2025) Real-time in vivo monitoring of cholinergic neurotransmission in the mouse brain using a microelectrochemical choline biosensor. Eur J Neurosci 62)9):e70291. doi: 10.1111/ejn.70291 PMID: 41185145
Objective: To refine an established choline oxidase (ChOx) microelectrochemical biosensor to validate its use for long-term recording in the freely moving mouse.
Summary: Systemic administration of donepezil produced a pronounced decrease in current in both the pre-frontal cortex and hippocampus, with scopolamine and amphetamine resulting in signal increases that were not observed in animals with selective saporin lesioning (mu p75-SAP) of the cholinergic basal forebrain. Furthermore, continuous biosensor recording in both regions displayed diurnal oscillations across repetitive light–dark phases. All are consistent with successful monitoring of endogenous changes in cholinergic neurotransmission.
Usage: Two 1-μL icv injections of either sterile PBS (control animals) or mu p75-SAP (IT-16) at a concentration of 0.6 μg/μL were made into the lateral ventricles usinga NanoFil syringe under the control of an infusion pump at a rate of 0.2 nL/min. Following injection, the needle tip was left in place for 8 min to minimize reflux.
Objective: To develop and validate a modular streptavidin based antibody drug conjugate platform for optimizing antibody mediated hematopoietic stem cell transplant conditioning.
Summary: The authors demonstrate that a streptavidin drug conjugate system enables rapid comparison of antibody payload combinations for hematopoietic stem cell depletion and leukemia targeting. The study builds on prior work using CD45 targeted immunotoxins to achieve effective hematopoietic niche depletion in murine HSCT models.
Usage: This study references earlier conditioning experiments that utilized Streptavidin-ZAP (IT-27) in combination with biotinylated CD45 antibodies to selectively deplete hematopoietic stem cells in murine HSCT models, typically administered as a single intravenous dose of approximately 75 µg per mouse.
Leanza MH, Storelli E, D’Arco D, de Leo G, Kleiner G, Arancio L, Capodieci G, Gulino R, Bava A, Leanza G (2025) Cerebellar contributions to spatial learning and memory: Effects of discrete immunotoxic lesions. Int J Mol Sci 26(19):9553. doi: 10.3390/ijms26199553 PMID: 41096819
Objective: To analyze the behavioral and anatomical effects of discrete injections, in different groups of animals, of the same 192 IgG-saporin toxin into the basal forebrain nuclei and/or into the cerebellar vermis and hemispheres.
Summary: Authors administered, 192 IgG-saporin, selectively targeting cholinergic neurons in the basal forebrain and a subpopulation of cerebellar Purkinje cells, to adult rats bilaterally into the basal forebrain nuclei, the cerebellar cortices or both areas combined. The results suggest important functional interactions between the ascending regulatory inputs from the cerebellum and those arising in the basal forebrain nuclei that would act together to modulate the complex sensory–motor and cognitive processes required to control whole body movement in space.
Usage: (i) bilateral intraventricular injection of 192 IgG-saporin (ICV, n = 12); (ii) bilateral injections of 192 IgG-saporin into the basal forebrain nuclei (BF, n = 12); (iii) injections of 192 IgG-saporin into the cerebellar hemispheres and vermis (CBL, n = 12); and (iv) injections of 192 IgG-saporin into both the basal forebrain nuclei and cerebellar hemispheres and vermis (BF/CBL, n = 12).
Herrerias A, Oliverio A, Dvorácskó S, Thyagarajan A, Chedester L, Liu J, Cinar R, Iyer MR, Kunos G, Godlewski G (2025) CB1 receptors on a subset of vagal afferent neurons modulate voluntary ethanol intake in mice. Mol Psychiatry doi: 10.1038/s41380-025-03266-9 PMID: 40975751
Objective: To investigate how peripheral CB1 receptors on vagal afferent neurons regulate voluntary ethanol intake and their role in gut-brain signaling underlying alcohol consumption.
Summary: Selective deletion or ablation of CB1R in nodose ganglion neurons abolished the inhibitory effects of peripheral CB1R antagonists on voluntary ethanol intake. The study identifies CB1R on Gpr65⁺ vagal sensory neurons as critical mediators of gut-brain communication regulating alcohol preference and intake.
Usage: Anti-CB1-SAP (IT-104) or Blank-SAP (IT-21) was injected into the proximity of the nodose ganglion at 250 ng/mL to selectively ablate CB1R-expressing vagal afferent neurons
Kobayashi K, Miyazaki Y, Sakamoto N, Yamamoto M, Nagat N, Murata T (2025) Long-term scratching analysis of mice using machine learning. PNAS Nexus pgaf292. doi: 10.1093/pnasnexus/pgaf292
Objective: To objectively quantify mouse scratching behavior across light and dark cycles using automated, long-term machine learning analysis.
Summary: Naïve BALB/c mice exhibited more frequent and persistent scratching during the light period, and DNFB-induced dermatitis caused biphasic, long-lasting scratching even during rest. The study establishes a continuous behavior analysis method that reveals circadian and pathological pruritus features.
Usage: Bombesin-SAP (IT-40) or Blank-SAP (IT-21) was administered intrathecally at 400 ng in 5 μL PBS.
