The role of the Ventral Nucleus of the Trapezoid Body in the auditory prepulse inhibition of the acoustic startle reflex.

Cholinergic signaling is essential to mediate the auditory prepulse inhibition (PPI), an operational measure of sensorimotor gating, that refers to the reduction of the acoustic startle reflex (ASR) when a low-intensity, non-startling acoustic stimulus (the prepulse) is presented just before the onset of the acoustic startle stimulus. The cochlear root neurons (CRNs) are the first cells of the ASR circuit to receive cholinergic inputs from non-olivocochlear neurons of the ventral nucleus of the trapezoid body (VNTB) and subsequently decrease their neuronal activity in response to auditory prepulses. Yet, the contribution of the VNTB-CRNs pathway to the mediation of PPI has not been fully elucidated. In this study, we used the immunotoxin anti-choline acetyltransferase (ChAT)-saporin as well as electrolytic lesions of the medial olivocochlear bundle to selectively eliminate cholinergic VNTB neurons, and then assessed the ASR and PPI paradigms. Retrograde track-tracing experiments were conducted to precisely determine the site of lesioning VNTB neurons projecting to the CRNs. Additionally, the effects of VNTB lesions and the integrity of the auditory pathway were evaluated via auditory brain responses tests, ChAT- and FOS-immunohistochemistry. Consequently, we established three experimental groups: 1) intact control rats (non-lesioned), 2) rats with bilateral lesions of the olivocochlear bundle (OCB-lesioned), and 3) rats with bilateral immunolesions affecting both the olivocochlear bundle and the VNTB (OCB/VNTB-lesioned). All experimental groups underwent ASR and PPI tests at several interstimulus intervals before the lesion and 7, 14, and 21 days after it. Our results show that the ASR amplitude remained unaffected both before and after the lesion across all experimental groups, suggesting that the VNTB does not contribute to the ASR. The%PPI increased across the time points of evaluation in the control and OCB-lesioned groups but not in the OCB/VNTB-lesioned group. At the ISI of 50 ms, the OCB-lesioned group exhibited a significant increase in%PPI (p < 0.01), which did not occur in the OCB/VNTB-lesioned group. Therefore, the ablation of cholinergic non-olivocochlear neurons in the OCB/VNTB-lesioned group suggests that these neurons contribute to the mediation of auditory PPI at the 50 ms ISI through their cholinergic projections to CRNs. Our study strongly reinforces the notion that auditory PPI encompasses a complex mechanism of top-down cholinergic modulation, effectively attenuating the ASR across different interstimulus intervals within multiple pathways.

Mitigating the Functional Deficit after Neurotoxic Motoneuronal Loss by an Inhibitor of Mitochondrial Fission.

Amyotrophic lateral sclerosis (ALS) is an extremely complex neurodegenerative disease involving different cell types, but motoneuronal loss represents its main pathological feature. Moreover, compensatory plastic changes taking place in parallel to neurodegeneration are likely to affect the timing of ALS onset and progression and, interestingly, they might represent a promising target for disease-modifying treatments. Therefore, a simplified animal model mimicking motoneuronal loss without the other pathological aspects of ALS has been established by means of intramuscular injection of cholera toxin-B saporin (CTB-Sap), which is a targeted neurotoxin able to kill motoneurons by retrograde suicide transport. Previous studies employing the mouse CTB-Sap model have proven that spontaneous motor recovery is possible after a subtotal removal of a spinal motoneuronal pool. Although these kinds of plastic changes are not enough to counteract the functional effects of the progressive motoneuron degeneration, it would nevertheless represent a promising target for treatments aiming to postpone ALS onset and/or delay disease progression. Herein, the mouse CTB-Sap model has been used to test the efficacy of mitochondrial division inhibitor 1 (Mdivi-1) as a tool to counteract the CTB-Sap toxicity and/or to promote neuroplasticity. The homeostasis of mitochondrial fission/fusion dynamics is indeed important for cell integrity, and it could be affected during neurodegeneration. Lesioned mice were treated with Mdivi-1 and then examined by a series of behavioral test and histological analyses. The results have shown that the drug may be capable of reducing functional deficits after the lesion and promoting synaptic plasticity and neuroprotection, thus representing a putative translational approach for motoneuron disorders.

Novel loading protocol combines highly efficient encapsulation of exogenous therapeutic toxin with preservation of extracellular vesicles properties, uptake and cargo activity.

Extracellular vesicles (EVs) have mostly been investigated as carriers of biological therapeutics such as proteins and RNA. Nevertheless, small-molecule drugs of natural or synthetic origin have also been loaded into EVs, resulting in an improvement of their therapeutic properties. A few methods have been employed for EV cargo loading, but poor yield and drastic modifications of vesicles remain unsolved challenges. We tested a different strategy based on temporary pH alteration through incubation of EVs with alkaline sodium carbonate, which resulted in conspicuous exogenous molecule incorporation. In-depth characterization showed that vesicle size, morphology, composition, and uptake were not affected. Our method was more efficient than gold-standard electroporation, particularly for a potential therapeutic toxin: the plant Ribosome Inactivating Protein saporin. The encapsulated saporin resulted protected from degradation, and was efficiently conveyed to receiving cancer cells and triggered cell death. EV-delivered saporin was more cytotoxic compared to the free toxin. This approach allows both the structural preservation of vesicle properties and the transfer of protected cargo in the context of drug delivery.

The basal forebrain cholinergic system as target for cell replacement therapy in Parkinson's disease.

In recent years there has been a renewed interest in the basal forebrain cholinergic system as a target for the treatment of cognitive impairments in patients with Parkinson's disease, due in part to the need to explore novel approaches to treat the cognitive symptoms of the disease and in part to the development of more refined imaging tools that have made it possible to monitor the progressive changes in the structure and function of the basal forebrain system as they evolve over time. In parallel, emerging technologies allowing the derivation of authentic basal forebrain cholinergic neurons from human pluripotent stem cells are providing new powerful tools for the exploration of cholinergic neuron replacement in animal models of Parkinson's disease-like cognitive decline. In this review, we discuss the rationale for cholinergic cell replacement as a potential therapeutic strategy in Parkinson's disease and how this approach can be explored in rodent models of Parkinson's disease-like cognitive decline, building on insights gained from the extensive animal experimental work that was performed in rodent and primate models in the 1980s and 90s. Although therapies targeting the cholinergic system have so far been focused mainly on patients with Alzheimer's disease, Parkinson's disease with dementia may be a more relevant condition. In Parkinson's disease with dementia, the basal forebrain system undergoes progressive degeneration and the magnitude of cholinergic cell loss has been shown to correlate with the level of cognitive impairment. Thus, cell therapy aimed to replace the lost basal forebrain cholinergic neurons represents an interesting strategy to combat some of the major cognitive impairments in patients with Parkinson's disease dementia.

The Induction of Long-Term Potentiation by Medial Septum Activation under Urethane Anesthesia Can Alter Gene Expression in the Hippocampus.

We studied changes in the expression of early genes in hippocampal cells in response to stimulation of the dorsal medial septal area (dMSA), leading to long-term potentiation in the hippocampus. Rats under urethane anesthesia were implanted with stimulating electrodes in the ventral hippocampal commissure and dMSA and a recording electrode in the CA1 area of the hippocampus. We found that high-frequency stimulation (HFS) of the dMSA led to the induction of long-term potentiation in the synapses formed by the ventral hippocampal commissure on the hippocampal CA1 neurons. One hour after dMSA HFS, we collected the dorsal and ventral hippocampi on both the ipsilateral (damaged by the implanted electrode) and contralateral (intact) sides and analyzed the expression of genes by qPCR. The dMSA HFS led to an increase in the expression of bdnf and cyr61 in the ipsilateral hippocampi and egr1 in the ventral contralateral hippocampus. Thus, dMSA HFS under the conditions of degeneration of the cholinergic neurons in the medial septal area prevented the described increase in gene expression. The changes in cyr61 expression appeared to be dependent on the muscarinic M1 receptors. Our data suggest that the induction of long-term potentiation by dMSA activation enhances the expression of select early genes in the hippocampus.

New Insights on Saporin Resistance to Chemical Derivatization with Heterobifunctional Reagents.

Saporin is a type 1 ribosome-inactivating protein widely used as toxic payload in the construction of targeted toxins, chimeric molecules formed by a toxic portion linked to a carrier moiety. Among the most used carriers, there are large molecules (mainly antibodies) and small molecules (such as neurotransmitters, growth factors and peptides). Some saporin-containing targeted toxins have been used for the experimental treatment of several diseases, giving very promising results. In this context, one of the reasons for the successful use of saporin lies in its resistance to proteolytic enzymes and to conjugation procedures. In this paper, we evaluated the influence of derivatization on saporin using three heterobifunctional reagents, namely 2-iminothiolane (2-IT), N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and 4-succinimidyloxycarbonyl-α-methyl-α-[2-pyridyldithio]toluene (SMPT). In order to obtain the highest number of inserted -SH groups with the lowest reduction of saporin biological activities, we assessed the residual ability of saporin to inhibit protein synthesis, to depurinate DNA and to induce cytotoxicity after derivatization. Our results demonstrate that saporin maintains an excellent resistance to derivatization processes, especially with SPDP, and permit us to define reaction conditions, in which saporin biological properties may not be altered. Therefore, these findings provide useful information for the construction of saporin-based targeted toxins, especially with small carriers.

Streptavidin-Saporin: Converting Biotinylated Materials into Targeted Toxins.

Streptavidin-Saporin can be considered a type of 'secondary' targeted toxin. The scientific community has taken advantage of this conjugate in clever and fruitful ways using many kinds of biotinylated targeting agents to send saporin into a cell selected for elimination. Saporin is a ribosome-inactivating protein that causes inhibition of protein synthesis and cell death when delivered inside a cell. Streptavidin-Saporin, mixed with biotinylated molecules to cell surface markers, results in powerful conjugates that are used both in vitro and in vivo for behavior and disease research. Streptavidin-Saporin harnesses the 'Molecular Surgery' capability of saporin, creating a modular arsenal of targeted toxins used in applications ranging from the screening of potential therapeutics to behavioral studies and animal models. The reagent has become a well-published and validated resource in academia and industry. The ease of use and diverse functionality of Streptavidin-Saporin continues to have a significant impact on the life science industry.

Expanding role of ribosome-inactivating proteins: From toxins to therapeutics.

Ribosome-inactivating proteins (RIPs) are toxic proteins with N-glycosidase activity. RIPs exert their action by removing a specific purine from 28S rRNA, thereby, irreversibly inhibiting the process of protein synthesis. RIPs can target both prokaryotic and eukaryotic cells. In bacteria, the production of RIPs aid in the process of pathogenesis whereas, in plants, the production of these toxins has been attributed to bolster defense against insects, viral, bacterial and fungal pathogens. In recent years, RIPs have been engineered to target a particular cell type, this has fueled various experiments testing the potential role of RIPs in many biomedical applications like anti-viral and anti-tumor therapies in animals as well as anti-pest agents in engineered plants. In this review, we present a comprehensive study of various RIPs, their mode of action, their significance in various fields involving plants and animals. Their potential as treatment options for plant infections and animal diseases is also discussed.

Quantum Dot-Based Screening Identifies F3 Peptide and Reveals Cell Surface Nucleolin as a Therapeutic Target for Rhabdomyosarcoma.

Active drug delivery by tumor-targeting peptides is a promising approach to improve existing therapies for rhabdomyosarcoma (RMS), by increasing the therapeutic effect and decreasing the systemic toxicity, e.g., by drug-loaded peptide-targeted nanoparticles. Here, we tested 20 different tumor-targeting peptides for their ability to bind to two RMS cell lines, Rh30 and RD, using quantum dots Streptavidin and biotin-peptides conjugates as a model for nanoparticles. Four peptides revealed a very strong binding to RMS cells: NCAM-1-targeting NTP peptide, nucleolin-targeting F3 peptide, and two Furin-targeting peptides, TmR and shTmR. F3 peptide showed the strongest binding to all RMS cell lines tested, low binding to normal control myoblasts and fibroblasts, and efficient internalization into RMS cells demonstrated by the cytoplasmic delivery of the Saporin toxin. The expression of the nucleophosphoprotein nucleolin, the target of F3, on the surface of RMS cell lines was validated by competition with the natural ligand lactoferrin, by colocalization with the nucleolin-binding aptamer AS1411, and by the marked sensitivity of RMS cell lines to the growth inhibitory nucleolin-binding N6L pseudopeptide. Taken together, our results indicate that nucleolin-targeting by F3 peptide represents a potential therapeutic approach for RMS.

RIPpore: A Novel Host-Derived Method for the Identification of Ricin Intoxication through Oxford Nanopore Direct RNA Sequencing.

Ricin is a toxin which enters cells and depurinates an adenine base in the sarcin-ricin loop in the large ribosomal subunit, leading to the inhibition of protein translation and cell death. We postulated that this depurination event could be detected using Oxford Nanopore Technologies (ONT) direct RNA sequencing, detecting a change in charge in the ricin loop. In this study, A549 cells were exposed to ricin for 2-24 h in order to induce depurination. In addition, a novel software tool was developed termed RIPpore that could quantify the adenine modification of ribosomal RNA induced by ricin upon respiratory epithelial cells. We provided demonstrable evidence for the first time that this base change detected is specific to RIP activity using a neutralising antibody against ricin. We believe this represents the first detection of depurination in RNA achieved using ONT sequencers. Collectively, this work highlights the potential for ONT and direct RNA sequencing to detect and quantify depurination events caused by ribosome-inactivating proteins such as ricin. RIPpore could have utility in the evaluation of new treatments and/or in the diagnosis of exposure to ricin.

Saporin Toxin Delivered by Engineered Colloidal Nanoparticles Is Strongly Effective against Cancer Cells.

Ribosome-inactivating proteins, including Saporin toxin, have found application in the search for innovative alternative cancer therapies to conventional chemo- and radiotherapy. Saporin's main mechanism of action involves the inhibition of cytoplasmic protein synthesis. Its strong theoretical efficacy is counterbalanced by negligible cell uptake and diffusion into the cytosol. In this work, we demonstrate that by immobilizing Saporin on iron oxide nanoparticles coated with an amphiphilic polymer, which promotes nanoconjugate endosomal escape, a strong cytotoxic effect mediated by ribosomal functional inactivation can be achieved. Cancer cell death was mediated by apoptosis dependent on nanoparticle concentration but independent of surface ligand density. The cytotoxic activity of Saporin-conjugated colloidal nanoparticles proved to be selective against three different cancer cell lines in comparison with healthy fibroblasts.

The potent α2-adrenoceptor antagonist RS 79948 also inhibits dopamine D2 -receptors: Comparison with atipamezole and raclopride.

Neurochemical, electrophysiological and behavioral evidence indicate that the potent α2-adrenoceptor antagonist RS 79948 is also a dopamine (DA) D2 receptor antagonist. Thus, results from ligand binding and adenylate cyclase activity indicate that RS 79948 binds to D2 receptors and antagonized D2 receptor-mediated inhibition of cAMP synthesis at nanomolar concentrations. Results from microdialysis indicated that RS 79948 shared with the selective α2-adrenergic antagonist atipamezole the ability to increase the co-release of DA and norepinephrine (NE) from noradrenergic terminals in the medial prefrontal cortex (mPFC), except that RS 79948-induced DA release persisted after noradrenergic denervation, unlike atipamezole effect, indicating that RS 79948 releases DA from dopaminergic terminals as well. Similarly to the D2 antagonist raclopride, but unlike atipamezole, RS 79948 increased extracellular DA and DOPAC in the caudate nucleus. Electrophysiological results indicate that RS 79948 shared with raclopride the ability to activate the firing of ventral tegmental area (VTA) DA neurons, while atipamezole was ineffective. Results from behavioral studies indicated that RS 79948 exerted effects mediated by independent, cooperative and contrasting inhibition of α2-and D2 receptors. Thus, RS 79948, but not atipamezole, prevented D2-autoreceptor mediated hypomotility produced by a small dose of quinpirole. RS 79948 potentiated, more effectively than atipamezole, quinpirole-induced motor stimulation. RS 79948 antagonized, less effectively than atipamezole, raclopride-induced catalepsy. Future studies should clarify if the dual α2-adrenoceptor- and D2-receptor antagonistic action might endow RS 79948 with potential therapeutic relevance in the treatment of schizophrenia, drug dependence, depression and Parkinson's disease.

Comparison of CD3e Antibody and CD3e-sZAP Immunotoxin Treatment in Mice Identifies sZAP as the Main Driver of Vascular Leakage.

Anti-CD3-epsilon (CD3e) monoclonal antibodies (mAbs) and CD3e immunotoxins (ITs) are promising targeted therapy options for various T-cell disorders. Despite significant advances in mAb and IT engineering, vascular leakage syndrome (VLS) remains a major dose-limiting toxicity for ITs and has been poorly characterized for recent "engineered" mAbs. This study undertakes a direct comparison of non-mitogenic CD3e-mAb (145-2C11 with Fc-silentTM murine IgG1: S-CD3e-mAb) and a new murine-version CD3e-IT (saporin-streptavidin (sZAP) conjugated with S-CD3e-mAb: S-CD3e-IT) and identifies their distinct toxicity profiles in mice. As expected, the two agents showed different modes of action on T cells, with S-CD3e-mAb inducing nearly complete modulation of CD3e on the cell surface, while S-CD3e-IT depleted the cells. S-CD3e-IT significantly increased the infiltration of polymorphonuclear leukocytes (PMNs) into the tissue parenchyma of the spleen and lungs, a sign of increased vascular permeability. By contrast, S-CD3e-mAbs-treated mice showed no notable signs of vascular leakage. Treatment with control ITs (sZAP conjugated with Fc-silent isotype antibodies) induced significant vascular leakage without causing T-cell deaths. These results demonstrate that the toxin portion of S-CD3e-IT, not the CD3e-binding portion (S-CD3e-mAb), is the main driver of vascular leakage, thus clarifying the molecular target for improving safety profiles in CD3e-IT therapy.

A Novel RGD-4C-Saporin Conjugate Inhibits Tumor Growth in Mouse Models of Bladder Cancer.

Although toxin may have some advantages compared to chemotherapeutic drugs in cancer therapy, e.g. a potent cytotoxic activity and a reduced risk of resistance, their successful application in the treatments to solid tumors still remains to be fully demonstrated. In this study, we genetically modified the structure of the plant-derived single-chain ribosome inactivating protein saporin (SAP) by fusing its N-terminus to the ACDCRGDCFCG peptide (RGD-4C), an αv-integrin ligand, and explored the anti-tumor activity of the resulting protein (called RGD-SAP) in vitro and in vivo, using a model of muscle invasive bladder cancer. We found that the RGD-4C targeting domain enhances the cytotoxic activity of SAP against various tumor cell lines, in a manner dependent on αv-integrin expression levels. In a subcutaneous syngeneic model of bladder cancer, RGD-SAP significantly reduced tumor growth in a dose-dependent manner. Furthermore, systemic administration of RGD-SAP in combination with mitomycin C, a chemotherapeutic drug currently used to treat patients with bladder cancer, increased the survival of mice bearing orthotopic bladder cancer with no evidence of systemic toxicity. Overall, the results suggest that RGD-SAP represents an efficient drug that could be exploited, either alone or in combination with the state-of-the-art therapies, for the treatment of bladder cancer and, potentially, of other solid tumors.

Spatio-Temporal Photoactivation of Cytotoxic Proteins.

Protein therapeutics offer exquisite selectivity in targeting cellular processes and behaviors, but are rarely used against non-cell surface targets due to their poor cellular uptake. While cell-penetrating peptides can be used to deliver recombinant proteins to the cytosol, it is generally difficult to selectively deliver active proteins to target cells. Here, we report a recombinantly produced, intracellular protein delivery and targeting platform that uses a photocaged intein to regulate the spatio-temporal activation of protein activity in selected cells upon irradiation with light. The platform was successfully demonstrated for two cytotoxic proteins to selectively kill cancer cells after photoactivation of intein splicing. This platform can generically be applied to any protein whose activity can be disrupted by a fused intein, allowing it to underpin a wide variety of future protein therapeutics.

Saporin as a Commercial Reagent: Its Uses and Unexpected Impacts in the Biological Sciences-Tools from the Plant Kingdom.

Saporin is a ribosome-inactivating protein that can cause inhibition of protein synthesis and causes cell death when delivered inside a cell. Development of commercial Saporin results in a technology termed 'molecular surgery', with Saporin as the scalpel. Its low toxicity (it has no efficient method of cell entry) and sturdy structure make Saporin a safe and simple molecule for many purposes. The most popular applications use experimental molecules that deliver Saporin via an add-on targeting molecule. These add-ons come in several forms: peptides, protein ligands, antibodies, even DNA fragments that mimic cell-binding ligands. Cells that do not express the targeted cell surface marker will not be affected. This review will highlight some newer efforts and discuss significant and unexpected impacts on science that molecular surgery has yielded over the last almost four decades. There are remarkable changes in fields such as the Neurosciences with models for Alzheimer's Disease and epilepsy, and game-changing effects in the study of pain and itch. Many other uses are also discussed to record the wide-reaching impact of Saporin in research and drug development.

Neurodegeneration in the centrally-projecting Edinger-Westphal nucleus contributes to the non-motor symptoms of Parkinson's disease in the rat.

BACKGROUND: The neuropathological background of major depression and anxiety as non-motor symptoms of Parkinson's disease is much less understood than classical motor symptoms. Although, neurodegeneration of the Edinger-Westphal nucleus in human Parkinson's disease is a known phenomenon, its possible significance in mood status has never been elucidated. In this work we aimed at investigating whether neuron loss and alpha-synuclein accumulation in the urocortin 1 containing (UCN1) cells of the centrally-projecting Edinger-Westphal (EWcp) nucleus is associated with anxiety and depression-like state in the rat. METHODS: Systemic chronic rotenone administration as well as targeted leptin-saporin-induced lesions of EWcp/UCN1 neurons were conducted. Rotarod, open field and sucrose preference tests were performed to assess motor performance and mood status. Multiple immunofluorescence combined with RNAscope were used to reveal the functional-morphological changes. Two-sample Student's t test, Spearman's rank correlation analysis and Mann-Whitney U tests were used for statistics. RESULTS: In the rotenone model, besides motor deficit, an anxious and depression-like phenotype was detected. Well-comparable neuron loss, cytoplasmic alpha-synuclein accumulation as well as astro- and microglial activation were observed both in the substantia nigra pars compacta and EWcp. Occasionally, UCN1-immunoreactive neuronal debris was observed in phagocytotic microglia. UCN1 peptide content of viable EWcp cells correlated with dopaminergic substantia nigra cell count. Importantly, other mood status-related dopaminergic (ventral tegmental area), serotonergic (dorsal and median raphe) and noradrenergic (locus ceruleus and A5 area) brainstem centers did not show remarkable morphological changes. Targeted partial selective EWcp/UCN1 neuron ablation induced similar mood status without motor symptoms. CONCLUSIONS: Our findings collectively suggest that neurodegeneration of urocortinergic EWcp contributes to the mood-related non-motor symptoms in toxic models of Parkinson's disease in the rat.

Serotonin-1A receptor activation in the median raphe nucleus improves response learning-based strategy in 192IgG saporin-induced cognitive impairments.

Deficits in the translation between egocentric-allocentric strategies may become another diagnostic mark for neurodegenerative disorders, especially Alzheimer's disease. Regarding the specific regional distribution of serotonin-1A receptor in brain areas mediating allocentric (externally-centered) spatial navigation to the escape location, here we studied the effects of median raphe nucleus serotonin-1A autoreceptors stimulation, [8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT); 4 µg/0.5 µl saline], of a selective cholinergic denervation by intracerebroventricular administration of the 192IgG saporin (1µl/each ventricle), on male Wistar rats search strategies in a Morris maze during acquisition, and before probe sessions. Despite some evidence of spatial hippocampal dependent knowledge to those PBS/Saline animals, their performance dropped to chance levels on probe trial. Therefore, we considered two probabilities and first analyzed the ability of the rats to make better use of one or more strategies. We showed statistically significant increases in the distances associated with egocentric (body-centered) non-spatial strategies, random searching in particular, in 192IgG/8OH rats, which led to their improved performance. Second, considering to what extent a shift in search strategy use improves performance indicated that 8-OH-DPAT alone did not affect learning since it appeared the related performance was impaired over days. However, the strategy choices made by 192IgG/8OH rats increased performance by more than 12% compared to 192IgG/Saline rats, an effect reversed with pre-treatment by serotonin-1A receptor antagonist, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane-carboxamide (WAY 100635). The results strongly suggest the potential role of serotonergic system, via the serotonin-1A receptors, in spatial navigation. We argue that the receptors are of interest as therapeutic targets that can be used against age-related cognitive decline.

Intrahippocampal Adeno-Associated Virus-Mediated Overexpression of Nerve Growth Factor Reverses 192IgG-Saporin-Induced Impairments of Hippocampal Plasticity and Behavior.

One of the aspects of Alzheimer disease is loss of cholinergic neurons in the basal forebrain, which leads to development of cognitive impairment. Here, we used a model of cholinergic deficit caused by immunotoxin 192IgG-saporin to study possible beneficial effects of adeno-associated virus (AAV)-mediated overexpression of nerve growth factor (NGF) in the hippocampus of rats with cholinergic deficit. Suspension of recombinant AAV carrying control cassette or cassette with NGF was injected into both hippocampi of control rats or rats with cholinergic deficit induced by intraseptal injection of 192IgG-saporin. Analysis of choline acetyltransferase (ChAT) immunostaining showed that NGF overexpression in the hippocampus did not prevent strong loss of ChAT-positive neurons in the septal area caused by the immunotoxin. Induction of cholinergic deficit in the hippocampus led to impairments in Y-maze and beam-walking test but did not affect behavioral indices in the T-maze, open field test, and inhibitory avoidance training. NGF overexpression in the rats with cholinergic deficit restored normal animal behavior in Y-maze and beam-walking test. Recording of field excitatory postsynaptic potentials in vivo in the hippocampal CA1 area showed that induction of cholinergic deficit decreased magnitude of long-term potentiation (LTP) and prevented a decrease in paired-pulse ratio after LTP induction, and NGF overexpression reversed these negative changes in hippocampal synaptic characteristics. The beneficial effect of NGF was not associated with compensatory changes in the number of cells that express NGF receptors TrkA and NGFR in the hippocampus and medial septal area. NGF overexpression also did not prevent a 192IgG-saporin-induced decrease in the activity of acetylcholine esterase in the hippocampus. We conclude that NGF overexpression in the hippocampus under conditions of cholinergic deficit induces beneficial effects which are not related to maintenance of cholinergic function.

Light-controlled elimination of PD-L1+ cells.

The programmed death ligand-1 (PD-L1), also known as CD274 or B7-H1, is mainly expressed on cancer cells and/or immunosuppressive cells in the tumor microenvironment (TME) and plays an essential role in tumor progression and immune escape. Immune checkpoint inhibitors (ICIs) of the PD-1/PD-L1 axis have shown impressive clinical success, however, the majority of the patients do not respond to immune checkpoint therapy (ICT). Thus, to overcome ICT resistance there is a high need for potent and novel strategies that simultaneously target both tumor cells and immunosuppressive cells in the TME. In this study, we show that the intracellular light-controlled drug delivery method photochemical internalization (PCI) induce specific and strongly enhanced cytotoxic effects of the PD-L1-targeting immunotoxin, anti-PD-L1-saporin (Anti-PDL1-SAP), in the PD-L1+ triple-negative breast cancer MDA-MB-231 cell line, while no enhanced efficacy was obtained in the PD-L1 negative control cell line MDA-MB-453. Using fluorescence microscopy, we reveal that the anti-PD-L1 antibody binds to PD-L1 on the surface of the MDA-MD-231 cells and overnight accumulates in late endosomes and lysosomes where it co-localizes with the PCI photosensitizer fimaporfin (TPCS2a). Moreover, light-controlled endosomal/lysosomal escape of the anti-PD-L1 antibody and fimaporfin into the cytosol was obtained. We also confirm that the breast MDA-MB-468 and the prostate PC-3 and DU-145 cancer cell lines have subpopulations with PD-L1 expression. In addition, we show that interferon-gamma strongly induce PD-L1 expression in the per se PD-L1 negative CT26.WT cells and enhance the PD-L1 expression in MC-38 cells, of which both are murine colon cancer cell lines. In conclusion, our work provides an in vitro proof-of-concept of PCI-enhanced targeting and eradication of PD-L1 positive immunosuppressive cells. This light-controlled combinatorial strategy has a potential to advance cancer immunotherapy and should be explored in preclinical studies.