Voltage-gated potassium channels play a crucial role in cellular repolarization and are potential therapeutic targets in neuroinflammatory disorders and neurodegenerative diseases. This study explores Tityus bahiensis scorpion venom for neuroactive peptides. We identified the αKtx12 peptide as a potent neuroprotective agent. In SH-SY5Y cells, αKtx12 significantly enhances viability, validating its pharmacological potential. And in the animal model, we elucidate central nervous system (CNS) mechanism of αKtx12 through neuroproteomic analyses highlighting αKtx12 as a valuable tool for characterizing neuroplasticity and neurotropism, revealing its ability to elicit more physiological responses. The peptide’s potential to promote cell proliferation and neuroprotection suggests a role in functional recovery from nervous system injury or disease. This research unveils the neuroactive potential of scorpion venom-derived αKtx12’s, offering insights into its pharmacological utility. The peptide’s impact on neuronal processes suggests a promising avenue for therapeutic development, particularly in neurodegenerative conditions.

Alzheimer's disease (AD) is a neurodegenerative disease with an unknown cause. Many studies have suggested that the imbalance between the clearance and accumulation of β-amyloid protein (Aβ) in the brain of AD patients is the main cause of AD development of AD. Meanwhile, drug transporters play a key role in the transport of drugs and endogenous substances in vivo as well as in the development of many diseases. Could they be related to the imbalance between Aβ clearance and accumulation? OATP1A2 is the most abundant subfamily of organic anion transporting polypeptides (OATPs) that transport amphipathic substrates. Its high bilateral expression in brain endothelial cells suggests it plays a crucial role in delivering drugs and neuroactive peptides to brain tissue. Could it also be involved in mediating the production and accumulation of Aβ in the central system? This could lead to an imbalance between Aβ clearance and accumulation, ultimately resulting in AD development. This hypothesis would be bold and novel in the field of science. In this study, we successfully established the OATP1A2-HEK293T transgenic cell model, and found that the uptake of Aβ1-42 by OATP1A2-HEK293T cells was significantly higher than that of NC-HEK293T control cells and human astrocytes by adding different concentrations of Aβ1-42 to the cells of each group, suggesting that OATP1A2 expressed in the human brain is involved in Aβ amyloid protein transport.

An extensive network of cutaneous nerves, neuropeptides, and specific receptors richly innervates the skin and influences a variety of physiological and pathological processes. The sensory and autonomic nerve fibers secrete a variety of neuropeptides that are essential to the different phases of wound healing. In addition to initiating a neurogenic inflammatory response in the early stages of healing, neuropeptides also control wound healing by influencing immune cells, repair cells, and the growth factor network. However, the precise mechanism by which they accomplish these roles in the context of cutaneous wound healing is still unknown. Investigating the mechanisms of action of neuropeptides in wound healing and potential therapeutic applications is therefore urgently necessary. The present review discusses the process of wound healing, types of neuropeptides, potential mechanisms underlying the role of neuropeptides in cutaneous wound healing, as well as some neuropeptide-derived treatment strategies, such as hydrogels, new dressings, electro stimulation, and skin-derived precursors. Future in-depth mechanistic studies of neuropeptides in cutaneous wound healing may provide opportunities to develop therapeutic technologies that harness the roles of neuropeptides in the wound healing process.

Pain emotion is a negative emotion caused by pain, which seriously affects the patients' physical and psychological health. The anterior cingulate cortex (ACC) is a key brain region in the occurrence and regulation of pain and painful emotion. In this paper, we summed up the relevant progress of researches on acupuncture improvement of pain emotion from neurotransmitters and receptors, cellular signaling pathways and neural circuits of ACC, in order to provide a basis for further research. 1) Acupuncture can regulate the long-term potentiation of ACC synaptic transmission by down-regulating the expression of N-methyl-D-aspartic acid receptor (NMDAR)； and up-regulating the expression of μ-, delta- and κ-opioid receptors, neuropeptide S (NPS)/ NPS receptor, and the number of parvalbumin-expressing interneurons to reduce neuronal excitability, relieving pain emotion； 2) Acupuncture can regulate synaptic transmission of ACC neurons by inhibiting protein kinase Mζ (PKMζ)-GluR1 and cAMP/PKA-ERK-CREB signaling pathways, and modulate synaptic plasticity to ameliorate pain emotion； 3) Acupuncture intervention induced inhibition of rostral ACCGlu-thalamic circuit may result in relief of pain emotion but not pain sensation, whereas inhibition of the rostral ACCGlu-ventrolateral periaqueductal gray matter(vlPAG) circuit may relieve pain sensation but not pain emotion. In addition, our team found that in the treatment of painful diseases by acupuncture, the addition of "spirit-regulating" acupoints can not only improve patients' negative emotion, but also significantly relive patients' pain sensation.

The neuropeptide S receptor (NPSR) has been identified as a potential therapeutic target for anxiety and post-traumatic stress disorder. Central administration of neuropeptide S (NPS) in male mice produces anxiolytic-like effects, hyperlocomotion, and memory enhancement. Currently, the literature is limited in the number of studies investigating the effects of NPS in female test subjects despite females facing a higher prevalence of anxiety-related pathology, as well as greater risk for adverse effects while taking psychoactive drugs. Moreover, no previous studies have considered the influence of estrous cycle on the effects of NPS. The present study investigates whether NPS-mediated behavioral phenotypes seen in males translate to females, and whether they are affected by estrous cycle stage. Female C57BL/6NCr mice were intracerebroventricularly cannulated and underwent behavioral paradigms to test locomotion, anxiety, and memory. Estrous cycle stage was determined through examination of vaginal cytology. Our results provide evidence that NPS-mediated behaviors are influenced by the estrous cycle. Administration of NPS decreased anxiety-like behaviors more robustly when the female mice were in high estrogen stages of the estrous cycle. Therefore, the desired anxiolytic-like effects of targeting the NPSR are intact in female mice. However, these effects may to be influenced by the stage of the estrous cycle. The NPSR remains a strong potential drug target for new anxiolytic compounds and based on our initial observations further studies exploring the interaction of estrous cycle and the NPS system are warranted. SIGNIFICANCE STATEMENT: The neuropeptide S (NPS) receptor has been identified as a potential target for treating anxiety, a condition that is most prevalent in females. Therefore, the potential interaction of estrous cycle with the NPS system described in the present study is an important first step in understanding the function of the NPS system in females.

Different populations of hypothalamic kisspeptin (KISS1) neurons located in the rostral periventricular area of the third ventricle (RP3V) and arcuate nucleus (ARC) are thought to generate the sex‐specific patterns of gonadotropin secretion. These neuronal populations integrate gonadal sex steroid feedback with internal and external cues relayed via the actions of neurotransmitters and neuropeptides. The excitatory amino acid neurotransmitter glutamate, the main excitatory neurotransmitter in the brain, plays a role in regulating gonadotropin secretion, at least partially through engaging KISS1 signaling. The expression and function of individual glutamate receptor subtypes in KISS1 neurons, however, are not well characterized. Here, we used GCaMP‐based calcium imaging and patch‐clamp electrophysiology to assess the impact of activating individual ionotropic (iGluR) and group I metabotropic (mGluR) glutamate receptors on KISS1 neuron activity in the mouse RP3V and ARC. Our results indicate that activation of all iGluR subtypes and of group I mGluRs, likely mGluR1, consistently drives activity in the majority of KISS1 neurons within the RP3V and ARC of males and females. Our results also revealed, somewhat unexpectedly, sex‐ and region‐specific differences. Indeed, activating (S)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) type iGluRs evoked larger responses in female ARCKISS1 neurons than in their male counterparts whereas activating group I mGluRs induced larger responses in RP3VKISS1 neurons than in ARCKISS1 neurons in females. Together, our findings suggest that glutamatergic neurotransmission in KISS1 neurons, and its impact on the activity of these cells, might be sex‐ and region‐dependent in mice.

Introduction: Silver nanoparticles have a diversity of applications both in biological and technological areas. More recently, studies conducted in the Nano/Bio interface have demonstrated that chiral nanocrystals grew in chiral templates, and nanostructures functionalized with chiral molecules present specific properties. These properties apply to advanced materials, energy, medicine, and pharmacology.Methodology: The present study synthesized silver nanoparticles on silver seeds using D- and L-histidine as templates and borohydride as a reducing agent.Results and Discussion: The nanoparticles were characterized by UV-visible spectroscopy and presented surface plasmon resonance (SPR) bands around 415 nm. CD spectra showed signals in the region of the SPR band, indicating the growth of nanocrystals with chiral distortion. Synthesized silver nanoparticles were also characterized by high-resolution transmission electron microscopy (HRTEM), which evidenced the presence of histidine corona. The silver nanoparticles were functionalized with prolyl-oligopeptidase (POP), a prolinespecific endopeptidase expressed in the brain. This enzyme cleaves neuroactive peptides involved in memory, learning, and neurodegeneration. The enzyme POP was expressed with a His-tag to provide competitive binding affinity to silver nanoparticles covered by D- and L-histidine. Considering the biological importance, POP was chosen as a model for studying the functionalization of chiral silver nanoparticles regarding the chiral discrimination for binding affinity and stabilization.

Neuropeptides (NPs) and their cognate receptors are critical effectors of diverse physiological processes and behaviors. We recently reported of a noncanonical function of the Drosophila Glucose-6-Phosphatase (G6P) gene in a subset of neurosecretory cells in the central nervous system that governs systemic glucose homeostasis in food-deprived flies. Here, we show that G6P-expressing neurons define six groups of NP-secreting cells, four in the brain and two in the thoracic ganglion. Using the glucose homeostasis phenotype as a screening tool, we find that neurons located in the thoracic ganglion expressing FMRFamide NPs (FMRFaG6P neurons) are necessary and sufficient to maintain systemic glucose homeostasis in starved flies. We further show that G6P is essential in FMRFaG6P neurons for attaining a prominent Golgi apparatus and secreting NPs efficiently. Finally, we establish that G6P-dependent FMRFa signaling is essential for the build-up of glycogen stores in the jump muscle which expresses the receptor for FMRFamides. We propose a general model in which the main role of G6P is to counteract glycolysis in peptidergic neurons for the purpose of optimizing the intracellular environment best suited for the expansion of the Golgi apparatus, boosting release of NPs and enhancing signaling to respective target tissues expressing cognate receptors.

Linked rhythmic behaviors, such as respiration/locomotion or swallowing/chewing often require coordination for proper function. Despite its prevalence, the cellular mechanisms controlling coordination of the underlying neural networks remain undetermined in most systems. We use the stomatogastric nervous system of the crab Cancer borealis to investigate mechanisms of inter-network coordination, due to its small, well characterized feeding-related networks (gastric mill [chewing, ∼0.1 Hz]; pyloric [filtering food, ∼1 Hz]). Here, we investigate coordination between these networks during the Gly1-SIFamide neuropeptide modulatory state. Gly1-SIFamide activates a unique triphasic gastric mill rhythm in which the typically pyloric-only LPG neuron generates dual pyloric- plus gastric mill-timed oscillations. Additionally, the pyloric rhythm exhibits shorter cycles during gastric mill rhythm-timed LPG bursts, and longer cycles during IC, or IC plus LG gastric mill neuron bursts. Photoinactivation revealed that LPG is necessary to shorten pyloric cycle period, likely through its rectified electrical coupling to pyloric pacemaker neurons. Hyperpolarizing current injections demonstrated that although LG bursting enables IC bursts, only gastric mill rhythm bursts in IC are necessary to prolong the pyloric cycle period. Surprisingly, LPG photoinactivation also eliminated prolonged pyloric cycles, without changing IC firing frequency or gastric mill burst duration, suggesting that pyloric cycles are prolonged via IC synaptic inhibition of LPG, which indirectly slows the pyloric pacemakers via electrical coupling. Thus, the same dual-network neuron directly conveys excitation from its endogenous bursting and indirectly funnels synaptic inhibition to enable one network to alternately decrease and increase the cycle period of a related network.Significance Statement Related rhythmic behaviors frequently exhibit coordination, yet the cellular mechanisms coordinating the underlying neural networks are not determined in most systems. We investigated coordination between two small, well-characterized crustacean feeding-associated networks during a neuropeptide-elicited modulatory state. We find that a dual fast/slow network neuron directly shortens fast network cycles during its slow, intrinsically generated bursts, likely via electrical coupling to fast network pacemakers, despite rectification favoring the opposite direction. Additionally, the fast network is indirectly prolonged during another slow-network phase, via chemical synaptic inhibition that is likely funneled through the same electrical synapse. Thus, a dual-network neuron alternately reinforces and diminishes neuropeptide actions, enabling distinct frequencies of a faster network across different phases of a related slower rhythm.

Spexin (SPX, NPQ) is a 14-amino acid neuroactive peptide identified using bioinformatics. This amino acid sequence of the mature spexin peptide has been highly conserved during species evolution and is widely distributed in the central nervous system and peripheral tissues and organs. Therefore, spexin may play a role in various biological functions. Spexin, the cognate ligand for GALR2/3, acting as a neuromodulator or endocrine signaling factor, can inhibit reproductive performance. However, controversies and gaps in knowledge persist regarding spexin-mediated regulation of animal reproductive functions. This review focuses on the hypothalamic-pituitary-gonadal axis and provides a comprehensive overview of the impact of spexin on reproduction. Through this review, we aim to enhance understanding and obtain in-depth insights into the regulation of reproduction by spexin peptides, thereby providing a scientific basis for future investigations into the molecular mechanisms underlying the influence of spexin on reproductive function. Such investigations hold potential benefits for optimizing farming practices in livestock, poultry, and fish industries.

The advent of the fourth industrial revolution, characterized by artificial intelligence (AI) as its central component, has resulted in the mechanization of numerous previously labor-intensive activities. The use of in silico tools has become prevalent in the design of biopharmaceuticals. Upon conducting a comprehensive analysis of the genomes of many organisms, it has been discovered that their tissues can generate specific peptides that confer protection against certain diseases. This study aims to identify a selected group of neuropeptides (NPs) possessing favorable characteristics that render them ideal for production as neurological biopharmaceuticals. Until now, the construction of NP classifiers has been the primary focus, neglecting to optimize these characteristics. Therefore, in this study, the task of creating ideal NPs has been formulated as a multi-objective optimization problem. The proposed framework, NPpred, comprises two distinct components: NSGA-NeuroPred and BERT-NeuroPred. The former employs the NSGA-II algorithm to explore and change a population of NPs, while the latter is an interpretable deep learning-based model. The utilization of explainable AI and motifs has led to the proposal of two novel operators, namely p-crossover and p-mutation. An online application has been deployed at https://neuropred.anvil.app for designing an ideal collection of synthesizable NPs from protein sequences.

An insect neuroactive helix ring peptide called U11-MYRTX-Tb1a (abbreviated as U11) from the venom of the ant, Tetramorium bicarinatum. U11 is a 34-amino-acid peptide that is claimed to be one of the most paralytic peptides ever reported from ant venoms acting against blowflies and honeybees. The peptide features a compact triangular ring helix structure stabilized by a single disulfide bond, which is a unique three-dimensional scaffold among animal venoms. Pharmacological assays using Drosophila S2 cells have demonstrated that U11 is not cytotoxic but instead suggest that it may modulate potassium channels via the presence of a functional dyad. In our work described here, we have tested this hypothesis by investigating the action of synthetically made U11 on a wide array of voltage-gated K and Na channels since it is well known that these channels play a crucial role in the phenomenon of paralysis. Using the Xenopus laevis oocyte heterologous expression system and voltage clamp, our results have not shown any modulatory effect of 1 μM U11 on the activity of Kv1.1, Kv1.3, Kv1.4, Kv1.5, Shaker IR, Kv4.2, Kv7.1, Kv10.1, Kv11.1 and KQT1, nor on DmNav and BgNav. Instead, 10 μM U11 caused a quick and irreversible cytolytic effect, identical to the cytotoxic effect caused by Apis mellifera venom, which indicates that U11 can act as a pore-forming peptide. Interestingly, the paralytic dose (PD50) on blowflies and honeybees corresponds with the concentration at which U11 displays clear pore-forming activity. In conclusion, our results indicate that the insecticidal and paralytic effects caused by U11 may be explained by the putative pore formation of the peptide.

Neuropeptide S (NPS) was postulated to be a wake-promoting neuropeptide with unknown mechanism, and a mutation in its receptor (NPSR1) causes the short sleep duration trait in humans. We investigated the role of different NPS+ nuclei in sleep/wake regulation. Loss-of-function and chemogenetic studies revealed that NPS+ neurons in the parabrachial nucleus (PB) are wake-promoting, whereas peri-locus coeruleus (peri-LC) NPS+ neurons are not important for sleep/wake modulation. Further, we found that a NPS+ nucleus in the central gray of the pons (CGPn) strongly promotes sleep. Fiber photometry recordings showed that NPS+ neurons are wake-active in the CGPn and wake/REM-sleep active in the PB and peri-LC. Blocking NPS-NPSR1 signaling or knockdown of Nps supported the function of the NPS-NPSR1 pathway in sleep/wake regulation. Together, these results reveal that NPS and NPS+ neurons play dichotomous roles in sleep/wake regulation at both the molecular and circuit levels.

Placental corticotropin-releasing hormone (pCRH) is a neuroactive peptide produced in high concentrations in mid-late pregnancy, during key periods of fetal brain development. Some evidence suggests that higher pCRH exposure during gestation is associated with adverse neurodevelopment, particularly in female offspring. In 858 mother-child dyads from the sociodemographically diverse CANDLE cohort (Memphis, TN), we examined: (1) the slope of pCRH rise in mid-late pregnancy and (2) estimated pCRH at delivery as a measure of cumulative prenatal exposure. When children were 4 years-old, mothers reported on problem behaviors using the Child Behavior Checklist (CBCL) and cognitive performance was assessed by trained psychologists using the Stanford-Binet Intelligence Scales. We fitted linear regression models examining pCRH in relation to behavioral and cognitive performance measures, adjusting for covariates. Using interaction models, we evaluated whether associations differed by fetal sex, breastfeeding, and postnatal neighborhood opportunity. In the full cohort, log-transformed pCRH measures were not associated with outcomes; however, we observed sex differences in some models (interaction p-values≤0.01). In male offspring, an interquartile (IQR) increase in pCRH slope (but not estimated pCRH at delivery), was positively associated with raw Total (β=3.06, 95%CI: 0.40, 5.72), Internalizing (β=0.89, 95%CI: 0.03, 1.76), and Externalizing (β=1.25, 95%CI: 0.27, 2.22) Problem scores, whereas, in females, all associations were negative (Total Problems: β=−1.99, 95%CI: −3.89, −0.09; Internalizing: β=−0.82, 95%CI: −1.42, −0.23; Externalizing: β=−0.56, 95%CI: −1.34, 0.22). No associations with cognitive performance were observed nor did we observe moderation by breastfeeding or postnatal neighborhood opportunity. Our results provide further evidence that prenatal pCRH exposure may impact subsequent child behavior in sex-specific ways, however in contrast to prior studies suggesting adverse impacts in females, steeper mid-gestation pCRH rise was associated with more problem behaviors in males, but fewer in females.

Endometriosis is a chronic gynecological syndrome characterized by endometrial cell invasion of the extra-uterine milieu, pelvic pain and infertility. Treatment relies on either symptomatic drugs or hormonal therapies, even though the mechanism involved in the onset of endometriosis is yet to be elucidated. The signaling of sphingolipid sphingosine 1-phosphate (S1P) is profoundly dysregulated in endometriosis. Indeed, sphingosine kinase (SK)1, one of the two isoenzymes responsible for S1P biosynthesis, and S1P1, S1P3 and S1P5, three of its five specific receptors, are more highly expressed in endometriotic lesions compared to healthy endometrium. Recently, missense coding variants of the gene encoding the receptor 1 for neuropeptide S (NPS) have been robustly associated with endometriosis in humans. This study aimed to characterize the biological effect of NPS in endometriotic epithelial cells and the possible involvement of the S1P signaling axis in its action. NPS was found to potently induce cell invasion and actin cytoskeletal remodeling. Of note, the NPS-induced invasive phenotype was dependent on SK1 and SK2 as well as on S1P1 and S1P3, given that the biological action of the neuropeptide was fully prevented when one of the two biosynthetic enzymes or one of the two selective receptors was inhibited or silenced. Furthermore, the RhoA/Rho kinase pathway, downstream to S1P receptor signaling, was found to be critically implicated in invasion and cytoskeletal remodeling elicited by NPS. These findings provide new information to the understanding of the molecular mechanisms implicated in endometriosis pathogenesis, establishing the rationale for non-hormonal therapeutic targets for its treatment.

Schistosomes cause schistosomiasis, one of the neglected tropical diseases as defined by the WHO. For decades, the treatment of schistosomiasis relies on a single drug, praziquantel. Due to its wide use, there is justified fear of resistance against this drug, and a vaccine is not available. Besides its biological relevance in signal transduction processes, the class of G protein-coupled receptors (GPCRs) is also well suited for drug design. Against this background, we characterized one GPCR of Schistosoma mansoni, SmGPCR20, at the molecular and functional level. We identified two potential neuropeptides (NPPs) as ligands, SmNPP26 and SmNPP40, and unraveled their roles, in combination with SmGPCR20, in neuronal processes controlling egg production, oogenesis, and growth of S. mansoni females. Since eggs are closely associated with the pathogenesis of schistosomiasis, our results contribute to the understanding of processes leading to egg production in schistosomes, which is under the control of pairing in this exceptional parasite.

Anxiolytic-like effects of the dual orexin receptor antagonist daridorexant in rats

Background: Gouty arthritis causes severe pain and inflammation. Alginate oligosaccharides (AOSs) are natural products derived from alginate and have anti-inflammatory properties. We explored the potential effects of AOSs with different degrees of polymerization (Dp) on gouty arthritis and associated mechanisms. Methods: We established a mouse model of gouty arthritis by injecting monosodium urate (MSU) into ankle joint. Nocifensive behavior, gait and ankle swelling were used to study AOS's effects. Biochemical assays, in vivo imaging, live cell Ca2+ imaging, electrophysiology, RNA-sequencing, etc. were used for mechanism exploration. Results: AOS2 (Dp=2), AOS3 (Dp=3) and AOS4 (Dp=4) all inhibited ankle swelling, whereas AOS2&3 produced the most obvious analgesia on model mice. AOS3, which was picked for further evaluation, produced dose-dependent ameliorative effects on model mice. AOS3 reversed gait impairments but did not alter locomotor activity. AOS3 inhibited NLRP3 inflammasome activation and inflammatory cytokine up-regulation in ankle joint. AOS3 ameliorated MSU-induced oxidative stress and reactive oxygen species (ROS) production both in vivo and in vitro and reversed the impaired mitochondrial bioenergetics. AOS3 activated the Nrf2 pathway and promoted Nrf2 disassociation from Keap1-bound complex and Nrf2 nuclear translocation, thus facilitating antioxidant gene expression via Nrf2-dependent mechanism. Nrf2 gene deficiency abolished AOS3's ameliorative effects on pain, inflammation and oxidative stress in ankle joints of model mice. AOS3 reduced TRPV1 functional enhancement in DRG neurons and constrained neuroactive peptide release. Conclusions: AOS3 ameliorates gouty arthritis via activating Nrf2-dependent antioxidant signaling, resulting in suppression of ROS-mediated NLRP3 inflammasome activation and TRPV1 enhancement. AOS3 may be novel therapeutics for gouty arthritis.

A cluster of neuropeptide S neurons regulates breathing and arousal

Abstract BackgroundThe blood‐brain barrier (BBB) limits the therapeutic perspective for central nervous system (CNS) disorders such as Alzheimer’s disease. Interestingly, monoclonal antibodies generated against receptor‐mediated transcytosis receptors are able to transport therapeutic proteins across the BBB. Nanobodies, which are the variable domain isolated from camelid heavy‐chain only antibodies have also shown their potential to deliver therapeutics into the CNS. Here we aimed to expand the panel of nanobodies targeting other brain endothelial cell receptors.MethodCamelids were immunized with a pool of DNA encoding for several brain endothelial cell receptors. One target was chosen to validate the immunization and discovery approach, CD98hc, previously suggested to allow drug shuttling across the BBB. Nanobody libraries were selected on Chinese Hamster Ovarian (CHO) cells overexpressing the target of interest. Selected nanobodies were screened in vivo based on their potential to deliver into the CNS either a biologically active neuropeptide neurotensin to reduce body temperature, or a BACE1 inhibiting antibody that reduces the accumulation of Aβ1‐40 species.ResultDNA immunization followed by selections on CHO cells overexpressing the target of interest resulted in specific anti‐mouse CD98hc binders with affinities in the low nanomolar range. In vivo screening of mouse CD98hc nanobody binders revealed some discrepancy between the two methods used. Whereas none of the tested nanobodies fused to neurotensin was able to reduce body temperature, nanobodies fused to the anti‐BACE1 antibody 1A11 effectively reduced Aβ1‐40 levels in the brain. We suggest that the neurotensin approach, although having shown robustness with anti‐TfR nanobodies, might lack sensitivity when screening non‐TfR binders. We suggest BACE1 inhibition as a more robust model to validate CD98hc‐mediated transport across the BBB.ConclusionThis is a proof of concept study in which we have optimized our nanobody discovery approach to find nanobodies against BBB targets, and validated CD98hc as a receptor to target in order to shuttle drugs into the brain. In the future we will explore whether nanobodies raised against novel brain endothelial cell receptors could also improve brain penetration of biologics.