Angiotensin-converting enzymes as druggable features of psychiatric and neurodegenerative disorders.

The renin-angiotensin system (RAS) plays essential roles in maintaining peripheral cardiovascular homeostasis, with its potential roles in the brain only being recognized more recently. Angiotensin-I-converting enzyme (ACE) is the main component of the RAS, and it has been implicated in various disorders of the brain. ACE and other RAS components, including the related enzyme ACE2, angiotensin peptides and their respective receptors, can participate in the pathological state, as well as with potential to contribute to neuroprotection and/or to complement existing treatments for psychiatric illness. In this narrative review, we aimed to identify the main studies describing the functions of the RAS and ACEs in the brain and their association with brain disorders. These include neurodegenerative disorders such as Parkinson's and Alzheimer's diseases, psychiatric illnesses such as schizophrenia, bipolar disorder, and depression. We also discuss the possible association of a functional polymorphism of the ACE gene with these brain diseases and the relevance of the neuroprotective and anti-inflammatory properties of ACE inhibitors (ACEis) and angiotensin receptor blockers (ARBs). Based on this, we conclude that there is significant potential value to the inclusion of ACEis and/or ARBs as a novel integrated approach for the treatment of various disorders of the brain, and particularly for psychiatric illness.

Assessing the role of Ndel1 oligopeptidase activity in congenital Zika syndrome: Potential predictor of congenital syndrome endophenotype and treatment response.

Maternal infections are among the main risk factors for cognitive impairments in the offspring. Zika virus (ZIKV) can be transmitted vertically, causing a set of heterogeneous birth defects, such as microcephaly, ventriculomegaly and corpus callosum dysgenesis. Nuclear distribution element like-1 (Ndel1) oligopeptidase controls crucial aspects of cerebral cortex development underlying cortical malformations. Here, we examine Ndel1 activity in an animal model for ZIKV infection, which was associated with deregulated corticogenesis. We observed here a reduction in Ndel1 activity in the forebrain associated with the congenital syndrome induced by ZIKV isolates, in an in utero and postnatal injections of different inoculum doses in mice models. In addition, we observed a strong correlation between Ndel1 activity and brain size of animals infected by ZIKV, suggesting the potential of this measure as a biomarker for microcephaly. More importantly, the increase of interferon (IFN)-beta signaling, which was used to rescue the ZIKV infection outcomes, also recovered Ndel1 activity to levels similar to those of uninfected healthy control mice, but with no influence on Ndel1 activity in uninfected healthy control animals. Taken together, we demonstrate for the first time here an association of corticogenesis impairments determined by ZIKV infection and the modulation of Ndel1 activity. Although further studies are still necessary to clarify the possible role(s) of Ndel1 activity in the molecular mechanism(s) underlying the congenital syndrome induced by ZIKV, we suggest here the potential of monitoring the Ndel1 activity to predict this pathological condition at early stages of embryos or offspring development, during while the currently employed methods are unable to detect impaired corticogenesis leading to microcephaly. Ndel1 activity may also be possibly used to follow up the positive response to the treatment, such as that employing the IFN-beta that is able to rescue the ZIKV-induced brain injury.

Unraveiling the correlation among neurodevelopmental and inflammatory biomarkers in patients with chronic schizophrenia.

INTRODUCTION: Nuclear distribution element like-1 (Ndel1) is a cytosolic oligopeptidase, which was suggested as a potential biomarker of aberrant neurodevelopment and early stage of schizophrenia (SCZ). The involvement of Ndel1 in neurite outgrowth, neuronal migration and neurodevelopment was demonstrated. Moreover, Ndel1 cleaves neuropeptides, including the endogenous antipsychotic peptide neurotensin, and lower Ndel1 activity was reported in SCZ patients compared with healthy controls (HCs). Changes in brain-derived neurotrophic factor (BDNF) and inflammatory cytokines levels were also implicated in SCZ. OBJECTIVE: This preliminary study aimed to investigate the interactions between these immune and neurodevelopmental/neurotrophic biomarkers, namely BDNF and the recently identified SCZ biomarker Ndel1. RESULTS: We observed lower Ndel1 activity and IL-4 levels, and higher BDNF levels, in plasma of SCZ (N = 23) compared with HCs (N = 29). Interestingly, significant correlation between Ndel1 activity and IL-4 levels was observed in SCZ, while no correlation with any other evaluated interleukins (namely IL-2, IL-8, IL-10 and IL-17A) or BDNF levels was noticed. CONCLUSION: Although this hypothesis needs to be further explored for a better understanding of the mechanisms by which these altered pathways are associated to each other in SCZ, we suggest that Ndel1 and the inflammatory marker IL-4 are directly correlated.

Impact of Reck expression and promoter activity in neuronal in vitro differentiation.

Reck (REversion-inducing Cysteine-rich protein with Kazal motifs) tumor suppressor gene encodes a multifunctional glycoprotein which inhibits the activity of several matrix metalloproteinases (MMPs), and has the ability to modulate the Notch and canonical Wnt pathways. Reck-deficient neuro-progenitor cells undergo precocious differentiation; however, modulation of Reck expression during progression of the neuronal differentiation process is yet to be characterized. In the present study, we demonstrate that Reck expression levels are increased during in vitro neuronal differentiation of PC12 pheochromocytoma cells and P19 murine teratocarcinoma cells and characterize mouse Reck promoter activity during this process. Increased Reck promoter activity was found upon induction of differentiation in PC12 cells, in accordance with its increased mRNA expression levels in mouse in vitro models. Interestingly, Reck overexpression, prior to the beginning of the differentiation protocol, led to diminished efficiency of the neuronal differentiation process. Taken together, our findings suggest that increased Reck expression at early stages of differentiation diminishes the number of neuron-like cells, which are positive for the beta-3 tubulin marker. Our data highlight the importance of Reck expression evaluation to optimize in vitro neuronal differentiation protocols.

A study in first-episode psychosis patients: does angiotensin I-converting enzyme (ACE) activity associated with genotype predict symptoms severity reductions after treatment with the atypical antipsychotic risperidone?

BACKGROUND: Our previous studies showed increased angiotensin I-converting enzyme (ACE) activity in chronic schizophrenia (SCZ) patients compared to healthy control (HC) volunteers, and the relevance of combining ACE genotype and activity for predicting SCZ was suggested. METHODS: ACE activity was measured in plasma of ACE insertion/deletion (I/D) genotyped HC volunteers (N = 53) and antipsychotic-naïve first-episode psychosis (FEP) patients (N = 45), assessed at baseline (FEB-B) and also after 2-months (FEP-2M) of treatment with the atypical antipsychotic risperidone. RESULTS: ACE activity measurements showed significant differences among HC, FEP-B and FEP-2M groups (F = 5.356, df = 2, p = 0.005), as well as between HC and FEP-2M (post-hoc Tukey's multiple comparisons test, p = 0.004). No correlation was observed for ACE activity increases and symptom severity reductions in FEP as assessed by total PANSS (r = -0.131, p = 0.434). FEP subgrouped by ACE I/D genotype showed significant ACE activity increases, mainly in the DD genotype subgroup. No correlation between ACE activity and age was observed in FEP or HC groups separately (r = 0.210, p = 0.392), but ACE activity levels differences observed between these groups were influenced by age. CONCLUSIONS: The importance of measuring the ACE activity in blood plasma, associated to ACE I/D genotyping to support the follow-up of FEP patients did not show correlation with general symptoms amelioration in the present study. However, new insights into the influence of age and I/D genotype for ACE activity changes in FEP individuals upon treatment was demonstrated.

Insights into the Effects of Crack Abuse on the Human Metabolome Using a NMR Approach.

Approximately 255 million people consume illicit drugs every year, among which 18 million use cocaine. A portion of this drug is represented by crack, but it is difficult to estimate the number of users since most are marginalized. However, there are no recognized efficacious pharmacotherapies for crack-cocaine dependence. Inflammation and infection in cocaine users may be due to behavior adopted in conjunction with drug-related changes in the brain. To understand the metabolic changes associated with the drug abuse disorder and identify biomarkers, we performed a 1H NMR-based metabonomic analysis of 44 crack users' and 44 healthy volunteers' blood serum. The LDA model achieved 98% of accuracy. From the water suppressed 1H NMR spectra analyses, it was observed that the relative concentration of lactate was higher in the crack group, while long chain fatty acid acylated carnitines were decreased, which was associated with their nutritional behavior. Analyses of the aromatic region of CPMG 1H NMR spectra demonstrated histidine and tyrosine levels increased in the blood serum of crack users. The reduction of carnitine and acylcarnitines and the accumulation of histidine in the serum of the crack users suggest that histamine biosynthesis is compromised. The tyrosine level points to altered dopamine concentration.

Oral treatment with a rattlesnake native polypeptide crotamine efficiently inhibits the tumor growth with no potential toxicity for the host animal and with suggestive positive effects on animal metabolic profile.

The efficacy of crotamine as antitumoral was first demonstrated by daily intraperitoneal (IP) injections of low doses of this toxin in an animal model bearing melanoma tumors. Significant inhibition of tumor growth and increased lifespan of mice bearing tumor was also noticed after 21 consecutive days of this daily IP administration of crotamine. However, due to the limited acceptance of treatments by IP route in clinical conditions, herein, we evaluated the antitumor effect of this native polypeptide employing the oral route. The efficacy of crotamine in inhibiting the melanoma growth in vivo, even after passing through the gastrointestinal tract of the animal, was confirmed here. In addition, biochemical biomarkers and also histopathological analysis showed both the absence of any potential toxic effects in tissues or organs of the animal in which the highest accumulation of crotamine is expected. Interestingly, a reduction of weight gain was observed mainly in animals with tumor treated with crotamine by IP route, but not by oral administration. Albeit, oral administered crotamine was able to significantly decrease the body weight gain of healthy animals without tumor. Taking advantage of this same experimental animal models receiving crotamine by oral route, it was possible to show metabolic changes as the increased capacity of glucose clearance, which was accompanied by a reduction of the total cholesterol, and by increased high-density lipoprotein levels, both observed mainly in the absence of tumor. Triglycerides and low-density lipoprotein were also significantly decreased, but only in the absence of tumor. Taken together, these data suggest a clear trend for metabolic positive effects and mischaracterize unhealthy condition of animals, with or without tumors, treated with crotamine for 21 days. In addition, this study confirmed the efficacy of crotamine administered by oral route as antitumor agent, which besides the additional advantage of administration convenience and decreased risk of toxic effects, allowed the serendipitous observation of several positive metabolic effects on treated animals.

NDE1 and NDEL1 from genes to (mal)functions: parallel but distinct roles impacting on neurodevelopmental disorders and psychiatric illness.

NDE1 (Nuclear Distribution Element 1, also known as NudE) and NDEL1 (NDE-Like 1, also known as NudEL) are the mammalian homologues of the fungus nudE gene, with important and at least partially overlapping roles for brain development. While a large number of studies describe the various properties and functions of these proteins, many do not directly compare the similarities and differences between NDE1 and NDEL1. Although sharing a high degree structural similarity and multiple common cellular roles, each protein presents several distinct features that justify their parallel but also unique functions. Notably both proteins have key binding partners in dynein, LIS1 and DISC1, which impact on neurodevelopmental and psychiatric illnesses. Both are implicated in schizophrenia through genetic and functional evidence, with NDE1 also strongly implicated in microcephaly, as well as other neurodevelopmental and psychiatric conditions through copy number variation, while NDEL1 possesses an oligopeptidase activity with a unique potential as a biomarker in schizophrenia. In this review, we aim to give a comprehensive overview of the various cellular roles of these proteins in a "bottom-up" manner, from their biochemistry and protein-protein interactions on the molecular level, up to the consequences for neuronal differentiation, and ultimately to their importance for correct cortical development, with direct consequences for the pathophysiology of neurodevelopmental and mental illness.

Lipidomics, Biomarkers, and Schizophrenia: A Current Perspective.

Lipidomics is a lipid-targeted metabolomics approach aiming at comprehensive analysis of lipids in biological systems. Recent technological progresses in mass spectrometry, nuclear magnetic resonance spectroscopy, and chromatography have significantly enhanced the developments and applications of metabolic profiling of lipids in more complex biological samples. As many diseases reveal a notable change in lipid profiles compared with that of healthy people, lipidomics have also been broadly introduced to scientific research on diseases. Exploration of lipid biochemistry by lipidomics approach will not only provide insights into specific roles of lipid molecular species in health and disease, but it will also support the identification of potential biomarkers for establishing preventive or therapeutic approaches for human health. This chapter aims to illustrate how lipidomics can contribute for understanding the biological mechanisms inherent to schizophrenia and why lipids are relevant biomarkers of schizophrenia. The application of lipidomics in clinical studies has the potential to provide new insights into lipid profiling and pathophysiological mechanisms underlying schizophrenia. The future perspectives of lipidomics in mental disorders are also discussed herein.

A current snapshot of common genomic variants contribution in psychiatric disorders.

In the past decade, numerous advances were achieved in psychiatric genetics. Particularly, the genome wide association studies (GWAS) have contributed to uncovering new genes and pathways associated to psychiatric disorders (PDs). At the same time, with increasing sample sizes in the GWAS, the polygenic risk score (PRS) promoted an additional tool for identification and evaluation the genetic risk quantitatively in PDs. This concept review presents the state of the art GWAS analysis and PRS focusing on the genetic underpinnings of PDs. © 2016 Wiley Periodicals, Inc.

Increased Angiotensin I-Converting Enzyme Activity in Zika Virus-Infected Mouse Brain: Implications for Therapy?

Congenital zika virus syndrome (CZS) has become a significant worldwide concern since the sudden rise of microcephaly related to zika virus (ZIKV) in Brazil. Primarily transmitted by Aedes mosquitoes, ZIKV shares serologic similarities with dengue virus (DENV), complicating the diagnosis and/or clinical management. The Angiotensin I-Converting Enzyme (ACE) was associated with either neuroprotective or anti-inflammatory properties in the central nervous system (CNS). The possible role(s) of ACE in these two flaviviruses infection remain largely unexplored. In this study, we evaluate ACE activity in the brain of ZIKV- or DENV-infected mice, both compared to MOCK, showing about 30% increased ACE activity only in ZIKV-infected mice (p = 0.024), while no change was noticed in brain from DENV-infected animals (p = 0.888). In addition, the treatment with interferon beta (IFNß), under conditions previously demonstrated to rescue the normal size of microcephalic brains determined by ZIKV infection, also restored ACE activity in ZIKV-infected animals to levels close to that of the MOCK control group. Although inflammatory responses expected for either ZIKV or DENV infections, only ZIKV was associated with microcephaly, as well as with increased ACE activity and reversion by treatment with IFNß. Furthermore, this increase in ACE activity was observed only after intracerebroventricular (ICV) injection (F (2, 16) = 7.907, p = 0.004), but not for intraperitoneal (IP) administration of ZIKV (F (2, 26) = 1.996, p = 0.156), suggesting that the observed central ACE activity modulation may be associated with the presence of this specific flavivirus in the brain.

Embryonic origin of two ASD subtypes of social symptom severity: the larger the brain cortical organoid size, the more severe the social symptoms.

BACKGROUND: Social affective and communication symptoms are central to autism spectrum disorder (ASD), yet their severity differs across toddlers: Some toddlers with ASD display improving abilities across early ages and develop good social and language skills, while others with "profound" autism have persistently low social, language and cognitive skills and require lifelong care. The biological origins of these opposite ASD social severity subtypes and developmental trajectories are not known. METHODS: Because ASD involves early brain overgrowth and excess neurons, we measured size and growth in 4910 embryonic-stage brain cortical organoids (BCOs) from a total of 10 toddlers with ASD and 6 controls (averaging 196 individual BCOs measured/subject). In a 2021 batch, we measured BCOs from 10 ASD and 5 controls. In a 2022 batch, we  tested replicability of BCO size and growth effects by generating and measuring an independent batch of BCOs from 6 ASD and 4 control subjects. BCO size was analyzed within the context of our large, one-of-a-kind social symptom, social attention, social brain and social and language psychometric normative datasets ranging from N = 266 to N = 1902 toddlers. BCO growth rates were examined by measuring size changes between 1- and 2-months of organoid development. Neurogenesis markers at 2-months were examined at the cellular level. At the molecular level, we measured activity and expression of Ndel1; Ndel1 is a prime target for cell cycle-activated kinases; known to regulate cell cycle, proliferation, neurogenesis, and growth; and known to be involved in neuropsychiatric conditions. RESULTS: At the BCO level, analyses showed BCO size was significantly enlarged by 39% and 41% in ASD in the 2021 and 2022 batches. The larger the embryonic BCO size, the more severe the ASD social symptoms. Correlations between BCO size and social symptoms were r = 0.719 in the 2021 batch and r = 0. 873 in the replication 2022 batch. ASD BCOs grew at an accelerated rate nearly 3 times faster than controls. At the cell level, the two largest ASD BCOs had accelerated neurogenesis. At the molecular level, Ndel1 activity was highly correlated with the growth rate and size of BCOs. Two BCO subtypes were found in ASD toddlers: Those in one subtype had very enlarged BCO size with accelerated rate of growth and neurogenesis; a profound autism clinical phenotype displaying severe social symptoms, reduced social attention, reduced cognitive, very low language and social IQ; and substantially altered growth in specific cortical social, language and sensory regions. Those in a second subtype had milder BCO enlargement and milder social, attention, cognitive, language and cortical differences. LIMITATIONS: Larger samples of ASD toddler-derived BCO and clinical phenotypes may reveal additional ASD embryonic subtypes. CONCLUSIONS: By embryogenesis, the biological bases of two subtypes of ASD social and brain development-profound autism and mild autism-are already present and measurable and involve dysregulated cell proliferation and accelerated neurogenesis and growth. The larger the embryonic BCO size in ASD, the more severe the toddler's social symptoms and the more reduced the social attention, language ability, and IQ, and the more atypical the growth of social and language brain regions.

Crotamine/siRNA Nanocomplexes for Functional Downregulation of Syndecan-1 in Renal Proximal Tubular Epithelial Cells.

Proteinuria drives progressive tubulointerstitial fibrosis in native and transplanted kidneys, mainly through the activation of proximal tubular epithelial cells (PTECs). During proteinuria, PTEC syndecan-1 functions as a docking platform for properdin-mediated alternative complement activation. Non-viral gene delivery vectors to target PTEC syndecan-1 could be useful to slow down alternative complement activation. In this work, we characterize a PTEC-specific non-viral delivery vector composed of the cell-penetrating peptide crotamine complexed with a syndecan-1 targeting siRNA. Cell biological characterization was performed in the human PTEC HK2 cell line, using confocal microscopy, qRT-PCR, and flow cytometry. PTEC targeting in vivo was carried out in healthy mice. Crotamine/siRNA nanocomplexes are positively charged, about 100 nm in size, resistant to nuclease degradation, and showed in vitro and in vivo specificity and internalization into PTECs. The efficient suppression of syndecan-1 expression in PTECs mediated by these nanocomplexes significantly reduced properdin binding (p < 0.001), as well as the subsequent complement activation by the alternative complement pathway (p < 0.001), as observed in either normal or activated tubular conditions. To conclude, crotamine/siRNA-mediated downregulation of PTEC syndecan-1 reduced the activation of the alternative complement pathway. Therefore, we suggest that the present strategy opens new venues for targeted proximal tubular gene therapy in renal diseases.

Sodium nitroprusside as an adjunctive treatment for schizophrenia reduces the Ndel1 oligopeptidase activity.

OBJECTIVE: Schizophrenia (SCZ) is a disabling disorder that continues to defy clinicians and researchers. We investigated the effects of sodium nitroprusside (sNP) in an animal model of SCZ and as an add-on therapy in patients and the relationship between treatment with sNP and activity of the nDel1 enzyme, whose involvement in the pathophysiology of the disorder has been suggested earlier. METHODS: Ndel1 activity was measured following sNP infusions in spontaneously hypertensive rats (SHR; 2.5 or 5.0 mg/kg) and in a double-blind trial with SCZ patients (0.5 µg/kg/min). RESULTS: Ndel1 activity was significantly reduced after sNP infusion in blood of SHR compared to controls, and in patients receiving sNP (t = 7.756, df = 97, p < 0.0001, dcohen = 1.44) compared to placebo. Reduced Ndel1 activity between baseline and the end of the infusion was only seen in patients after treatment with sNP. CONCLUSION: Our findings suggest that SCZ patients may benefit from adjunctive therapy with sNP and that the Ndel1 enzyme is a candidate biomarker of psychopathology in the disorder. Future research should look into the role of Ndel1 in SCZ and the potential effects of sNP and drugs with similar profiles of action in both animals and patients.

1H NMR Metabolomics and Lipidomics To Monitor Positive Responses in Children with Autism Spectrum Disorder Following a Guided Parental Intervention: A Pilot Study.

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition that is characterized by patients displaying at least two out of the classical symptoms, such as impaired social communication, impaired interactions, and restricted repetitive behavior. Early parent-mediated interventions, such as video modeling for parental training, were demonstrated to be a successful low-cost way to deliver care for children with ASD. Nuclear magnetic resonance (NMR)-based metabolomics/lipidomics has been successfully employed in several mental disorder studies. Metabolomics and lipidomics of 37 ASD patients (children, aged 3-8 years), who were divided into two groups, one control group with no parental-training intervention (N = 18) and the other in which the parents were trained by a video modeling intervention (ASD parental training, N = 19), were analyzed by proton NMR spectroscopy. Patients in the ASD parental-training group sera were seen to have increased glucose, myo-inositol, malonate, proline, phenylalanine, and gangliosides in their blood serum, while cholesterol, choline, and lipids were decreased, compared to the control group, who received no parental-training. Taken together, we demonstrated here significant changes in serum metabolites and lipids in ASD children, previously demonstrated to show clinical positive effects following a parental training intervention based on video modeling, delivered over 22 weeks. We demonstrate the value of applying metabolomics and lipidomics to identify potential biomarkers for clinical interventions follow-up in ASD.

Euthymic and depressed bipolar patients are characterized by different RNA editing patterns in blood.

Bipolar disorder (BD) is a worldwide leading cause of disability. Inflammation roles in this disease is well established. ADAR1-mediated RNA editing is one of the key mechanisms regulating the inflammatory response. We have identified a panel of RNA editing-based blood biomarkers which allowed to discriminate unipolar from BD depression with high accuracy. We confirmed here the diagnostic value of this panel in a new cohort of BD patients recruited in Brazil. We also identified new combinations which allow a clear discrimination of BD from healthy controls and among BD subgroups, confirming that RNA editing is a key mechanism in BD.

Identification of an ex vivo inhibitor of the schizophrenia biomarker Ndel1 by high throughput screening.

Ndel1 oligopeptidase activity shows promise as a potential biomarker for diagnosing schizophrenia (SCZ) and monitoring early-stage pharmacotherapy. Ndel1 plays a pivotal role in critical aspects of brain development, such as neurite outgrowth, neuronal migration, and embryonic brain formation, making it particularly relevant to neurodevelopmental disorders like SCZ. Currently, the most specific inhibitor for Ndel1 is the polyclonal anti-Ndel1 antibody (NOAb), known for its high specificity and efficient anti-catalytic activity. NOAb has been vital in measuring Ndel1 activity in humans and animal models, enabling the prediction of pharmacological responses to antipsychotics in studies with patients and animals. To advance our understanding of in vivo Ndel1 function and develop drugs for mental disorders, identifying small chemical compounds capable of specifically inhibiting Ndel1 oligopeptidase is crucial, including within living cells. Due to challenges in obtaining Ndel1's three-dimensional structure and its promiscuous substrate recognition, we conducted a high-throughput screening (HTS) of 2,400 small molecules. Nine compounds with IC50-values ranging from 7 to 56 µM were identified as potent Ndel1 inhibitors. Notably, one compound showed similar efficacy to NOAb and inhibited Ndel1 within living cells, although its in vivo use may pose toxicity concerns. Despite this, all identified compounds hold promise as candidates for further refinement through rational drug design, aiming to enhance their inhibitory efficacy, specificity, stability, and biodistribution. Our ultimate goal is to develop druggable Ndel1 inhibitors that can improve the treatment and support the diagnosis of psychiatric disorders like SCZ.

Aberrant phase separation is a common killing strategy of positively charged peptides in biology and human disease.

Positively charged repeat peptides are emerging as key players in neurodegenerative diseases. These peptides can perturb diverse cellular pathways but a unifying framework for how such promiscuous toxicity arises has remained elusive. We used mass-spectrometry-based proteomics to define the protein targets of these neurotoxic peptides and found that they all share similar sequence features that drive their aberrant condensation with these positively charged peptides. We trained a machine learning algorithm to detect such sequence features and unexpectedly discovered that this mode of toxicity is not limited to human repeat expansion disorders but has evolved countless times across the tree of life in the form of cationic antimicrobial and venom peptides. We demonstrate that an excess in positive charge is necessary and sufficient for this killer activity, which we name 'polycation poisoning'. These findings reveal an ancient and conserved mechanism and inform ways to leverage its design rules for new generations of bioactive peptides.

The natural cell-penetrating peptide crotamine targets tumor tissue in vivo and triggers a lethal calcium-dependent pathway in cultured cells.

Our goal was to demonstrate the in vivo tumor specific accumulation of crotamine, a natural peptide from the venom of the South American rattlesnake Crotalus durissus terrificus, which has been characterized by our group as a cell penetrating peptide with a high specificity for actively proliferating cells and with a concentration-dependent cytotoxic effect. Crotamine cytotoxicity has been shown to be dependent on the disruption of lysosomes and subsequent activation of intracellular proteases. In this work, we show that the cytotoxic effect of crotamine also involves rapid intracellular calcium release and loss of mitochondrial membrane potential as observed in real time by confocal microscopy. The intracellular calcium overload induced by crotamine was almost completely blocked by thapsigargin. Microfluorimetry assays confirmed the importance of internal organelles, such as lysosomes and the endoplasmic reticulum, as contributors for the intracellular calcium increase, as well as the extracellular medium. Finally, we demonstrate here that crotamine injected intraperitoneally can efficiently target remote subcutaneous tumors engrafted in nude mice, as demonstrated by a noninvasive optical imaging procedure that permits in vivo real-time monitoring of crotamine uptake into tumor tissue. Taken together, our data indicate that the cytotoxic peptide crotamine can be used potentially for a dual purpose: to target and detect growing tumor tissues and to selectively trigger tumor cell death.

A Native CPP from Rattlesnake with Therapeutic and Theranostic Properties.

The cell-penetrating peptides (CPPs) are characterized by the ability of internalization into cells in vitro and in vivo, and the ability of these peptides can rely on a high content of positive charges, as it is the case of the native CPP crotamine. Crotamine is a polypeptide of about 42 amino acid residues with high content of basic residues as Arg and Lys. Although most of known CPPs are linear peptides, native crotamine from the venom of a South American rattlesnake has a well-defined 3D structure stabilized by three disulfide bonds which guarantee the exposure of side chains of basic amino acids. This 3D structure also protects this amphipathic polypeptide from the degradation even if administered by oral route, therefore, protecting also the biological activities of crotamine. As several different biological properties of crotamine are dependent of cell penetration, the methods mainly employed for analyzing crotamine properties as anthelminthic and antimalarial activities, antimicrobial and antitumor activities, with a unique selective cytotoxic property against actively proliferating cells, as tumor cells, were chosen based on crotamine ability of internalization mediated by its positive charge. This native cationic polypeptide is also able to efficiently carry, with no need of covalent linkage with the cargo, genetic material into cells in vitro and in vivo, suggesting its use in gene therapy. Moreover, the possibility of decorating gold nanoparticles keeping the ability of transfecting cells was demonstrated. More recently, the ability of crotamine to interfere in animal metabolism, inducing browning of adipose tissue and increasing the energy expenditure, and its application in renal therapy was demonstrated. As crotamine also accumulates specifically in tumor cells in vivo, and the potential utility of crotamine as a theranostic agent was then suggested. Therefore, diverse methodologies employed for the characterization and exploration of the therapeutic applications of this promising native CPP for remediation of several pathogenic conditions are presented here.