Parkinson’s disease (PD) is a neurodegenerative disease that affects both motor and non-motor functions. Although motor impairment is a prominent clinical sign of PD, additional neurological symptoms may also occur, particularly in the preclinical and prodromal stages. Among these symptoms, social cognitive impairment is common and detrimental. This article aims to review non-motor symptoms in PD patients, focusing on social cognitive deficits. It also examines the specific characteristics of the CA2 region and its involvement in social behavior, highlighting recent advances and perspectives. Additionally, this review provides critical insights into and analysis of research conducted in rodents and humans, which may help improve the understanding of the current status of putative therapeutic strategies for social cognitive dysfunction in PD and potential avenues related to the function of the hippocampal CA2 region. Parkinson’s disease (PD) is a debilitating neurodegenerative disorder characterized by the loss of dopaminergic neurons in the pars compacta of the substantia nigra, a loss that is unfortunately irreversible . StudiesThe hippocampal formation (HF) is located within the medial temporal lobe in all mammalian species, close to the adjacent cerebral cortex, enabling its many crucial connections to various cortical regions. HF is a critical functional unit that contributes significantly to many vital cognitive processes in both humans and animals, including learning and memory , fear prCA2 exhibits unique morphological features, including a more loosely packed stratum pyramidal in comparison to CA1. In addition, pyramidal neurons in CA2 have an oval and dense soma, which is the largest among excitatory neurons within CA regions. Neurons in CA2 are also characterized by a hyperpolarized resting membrane potential and display specific action potential firing patterns . FurtherRecently, CA2 has been shown to play a central role in social behavior. Molecular markers such as Purkinje cell protein 4 (PCP4), a regulator of G protein signaling 14 protein (RGS14), and striatum-enriched protein–tyrosine phosphatase (STEP), help to identify the specific population of neurons in the CA2 region ,16,17,18Another test showed that AVPR1B knockout mice could not discriminate the object they explored and recognize its location like the control group . AVPR1B In summary, CA2 pyramidal neurons possess numerous distinctive morphological, physiological, and synaptic characteristics, as well as intrinsic and extrinsic connections that distinguish them from other CA regions see , and morPD is an irreversible neurodegenerative disease  characteIndeed, motor, autonomic, and cognitive impairments are commonly observed in PD . StudiesPatients with PD have impaired decision-making, as studies have shown that they have difficulty learning from feedback and making optimal decisions, possibly due to dopamine depletion in the basal ganglia . PD patiThe intricate alterations in social behavior observed in individuals with PD present a challenge in identifying precise molecular mechanisms or neural circuits, including the hippocampal CA2 region, that contribute to these changes. Further research is necessary to establish a correlation between CA2 functionality and specific social behavioral variations observed in PD patients. Nonetheless, a few studies have indicated a potential involvement of CA2 in human social behavior ,100,101.As the classical view of PD expands beyond its traditional characterization as a purely motor disorder, it becomes increasingly important to delve into the contributions of non-motor brain regions. Among these regions, the hippocampal formation has been widely studied, and findings indicate that the hippocampus may possess compensatory mechanisms that help mitigate cognitive impairments associated with PD . In paraThe presence of α-synuclein in brainstem nuclei serves as a defining feature of PD and is closely linked to motor impairment. However, it is worth noting that α-synuclein also accumulates in the cerebral cortex and hippocampus, where it plays a crucial role in the development of cognitive abilities ,47. ThisHowever, a recent study conducted in mice demonstrated that the infusion of α-synuclein fibrils into the CA2–CA3 region did not result in behavioral deficits or cell loss, despite the presence of α-synucleinopathy in this area . HoweverThe findings suggest that social cognitive impairments are present in both individuals with PD and animal models of the disease, potentially linked to CA2 function. However, the extent to which CA2 contributes to these cognitive deficits, such as social interaction and temporal ordering, remains uncertain. Furthermore, considering the structural disparities between rodents and humans , it is iThe treatment of PD involves various medications, but there is a notable lack of effective options for addressing non-motor symptoms, particularly social disorders. Therefore, it is crucial to identify treatments that can specifically target and alleviate these symptoms without exacerbating motor impairments. In this context, understanding the therapeutic potential of the CA2 region becomes highly relevant. Existing research has examined the effects of specific drugs on the CA2 region see . For exaIn summary, several medications hold potential for improving social memory, enhancing sociability, and reducing social anxiety. Among these is oxytocin, a hormone renowned for its role in promoting social bonding and trust. Studies have suggested that intranasal administration of oxytocin may improve social memory, increase sociability, and reduce social anxiety. Another class of medications, selective serotonin reuptake inhibitors (SSRIs), commonly prescribed as a treatment for depression and anxiety disorders, may also have a positive effect on social anxiety and social functioning. In addition, certain drugs that act as NMDA receptor antagonists, such as ketamine, have shown potential for improving sociability and reducing social anxiety, particularly in individuals with conditions such as autism spectrum disorder .Furthermore, the review of existing literature uncovers the potential impact of specific traditional Chinese medicines (TCMs) on the CA2 region. For example, administration of CS 4-O-sulfation increases the presence of perineuronal networks (PNNs) and excitatory–inhibitory synapses in CA2 . AnotherTo summarize the putative role of CA2 in the non-motor symptoms of PD, it is important to recognize that although PD is primarily known for its motor symptoms, cognitive impairment and emotional dysregulation are also prevalent. Animal studies have demonstrated that PD-like mice exhibited impaired performance in social behavioral tests, which may indicate some involvement of the CA2 region in social interaction deficits. Furthermore, postmortem studies have revealed CA2 neuronal loss in PD patients, suggesting the vulnerability of this region to neurodegenerative processes. These findings suggest that the CA2 region may play a role in the non-motor symptoms of PD, particularly in social cognition.


Previously, the discrimination of collagen types I and II was successfully achieved using peptide pitch angle and anisotropic parameter methods. However, these methods require fitting polarization second harmonic generation (SHG) pixel-wise information into generic mathematical models, revealing inconsistencies in categorizing collagen type I and II blend hydrogels. In this study, a ResNet approach based on multipolarization SHG imaging is proposed for the categorization and regression of collagen type I and II blend hydrogels at 0%, 25%, 50%, 75%, and 100% type II, without the need for prior time-consuming model fitting. A ResNet model, pretrained on 18 progressive polarization SHG images at 10° intervals for each percentage, categorizes the five blended collagen hydrogels with a mean absolute error (MAE) of 0.021, while the model pretrained on nonpolarization images exhibited 0.083 MAE. Moreover, the pretrained models can also generally regress the blend hydrogels at 20%, 40%, 60%, and 80% type II. In conclusion, the multipolarization SHG image-based ResNet analysis demonstrates the potential for an automated approach using deep learning to extract valuable information from the collagen matrix. Investigating collagen type I and II mixtures in osteoarthritis can provide insights into the underlying mechanisms of cartilage degeneration. The dynamic interrelationship among type I and type II collagen, in conjunction with other elements of the matrix and cellular functions, exerts a significant impact on maintaining the delicate equilibrium between the degradation and reparative processes within cartilages2. Second harmonic generation (SHG) microscopy is a powerful technique for visualizing 3D collagen fibers with exceptional resolution3. It utilizes the non-centrosymmetric molecular arrangements in biological tissues, such as collagen, myosin, tendons, and muscles, to generate second harmonic signals5. By employing polarization-resolved SHG (P-SHG) imaging, researchers can obtain detailed quantitative insights into sub-cellular collagen structures6. P-SHG imaging has gained significant interest in various medical disciplines, including breast cancer diagnosis6, skin irregularities assessment7, muscle characterization8, osteoarthritis diagnosis9, and detection of diseased tissues10. It allows for differentiation capabilities at the fiber-level collagen structures, providing valuable information for medical research and diagnostics. Various methods can be used for quantification and analysis of P-SHG specimens, including geometrical and statistical analysis, Fourier transform-based analysis, phasor approach, and grey-level co-occurrence matrix-based texture analysis. P-SHG imaging is particularly adept at capturing microscopic structural information with high spatial resolution and it directly visualizes the anisotropic nature of materials, revealing the orientation and alignment of molecular structures. The dipole moment of collagen is represented by a power series that includes the first hyperpolarizability term and this term describes the interaction of collagen with the incident electric field. Overall, P-SHG imaging offers unparalleled contrast and specificity in visualizing detailed collagen structures in tissues, making it an indispensable tool for understanding complex biological processes and advancing medical diagnostics12. However, P-SHG analysis for discriminating collagen types such as types I and II often stands on pixel-by-pixel calculation and involves manual efforts in data processing, computation time, parameter extraction, and interpretation of results. This subjectivity can introduce bias and may require domain expertise, making it less automated and potentially more time-consuming.Osteoarthritis, a degenerative joint disease, is associated with alterations in the collagen composition within the cartilage matrix14. DL models, although complex, can be more interpretable with appropriate techniques, such as visualization of feature maps, and can also be trained to handle noise and variations in data, making them more adaptable to real-world scenarios. Conventional P-SHG-based analysis has its strengths in investigating specific physical properties; however, DL analysis can offer advantages in terms of automation, generalization, scalability, and adaptability to various domains and large-scale datasets. One of the most significant advancements in the realm of convolutional neural networks (CNNs) is the introduction of ResNet  in 2015, which has gained significant popularity in various computer vision tasks, including image segmentation15. This architecture has since gained widespread attention for its deep learning exceptional ability to tackle the challenges of training very deep neural networks18. ResNet encourages each layer to learn the difference between the current input and the desired output, effectively learning only what is new and complex. This approach mitigates the vanishing gradient issue, allowing the model to be trained effectively even with dozens or hundreds of layers. Regression tasks involve predicting continuous numerical values, making them applicable in situations where precise quantification is crucial19. In the context of biomedical research and medical imaging, regression models can be particularly valuable in quantifying the properties of tissues, organs, or other biological structures21. When adapting ResNet for regression tasks, the architecture's residual blocks can be modified to suit the nature of the prediction problem. The final fully connected layers are adjusted to produce a single continuous output instead of class probabilities. By integrating polarization SHG images into the ResNet architecture for regression tasks, the benefits of both modalities can be exploited. During the training process, ResNet learns to map the input polarization SHG images to the corresponding target values, effectively learning the complex relationships between polarization patterns and the underlying sample properties.Deep learning (DL) has revolutionized various domains in computer vision, achieving remarkable success in image classification, object detection, and segmentation tasks22. Another study used rheological analysis to conclude that the ratio of collagen type I to collagen type II affects the protein concentrations and collagen incorporation in the gels23. Some studies have also quantified the structural information of fiber networks in hydrogels using SHG tensor element ratios and fiber thickness purely from polarization-based SHG images where the model is limited to a pixelized method of assuming fiber of one-pixel width24. In this study, the application of progressive multipolarization SHG images incorporated in a multichannel 2D ResNet is explored for the discrimination of different collagen type I and II blend hydrogels. Herein, the multipolarization SHG images refer to the set of 18 optical cross-sectional SHG images obtained by incrementally rotating the linear polarization angle in 10° intervals across 180°. Nonpolarization SHG images are generated by averaging the intensities of the 18 linear polarization images, effectively resulting in an image devoid of any polarization change. This ensures a consistent laser polarization approach for both these images, considering the inherent random alignment of collagen fibers within the sample. The ResNet regression is trained with both the multipolarization and the nonpolarization images for premixing five hydrogels with the collagen type I and II blend hydrogel composition at (a) 0%, (b) 25%, (c) 50%, (d) 75%, and (e) 100% type II. Furthermore, this trained model is utilized to predict the blend hydrogels at 20%, 40%, 60%, and 80% type II. The experimental results confirm the efficacy of incorporating both modalities into categorization and regression analysis of collagen type I and II blend hydrogel. These findings offer valuable insights that can contribute to an enhanced understanding of collagen dynamics in the extracellular matrix (ECM) during cartilage repair.Recently, cartilage tissue engineering has emerged as a promising field in regenerative medicine, aiming to address the challenges associated with cartilage repair and regeneration. To overcome the limitations of traditional treatments, researchers have explored the development of biomimetic scaffolds that mimic the native extracellular matrix (ECM) to support cell growth and tissue regeneration. Among these scaffolds, collagen-based hydrogels have shown great potential due to their biocompatibility, biodegradability, and similarity to the natural ECM of cartilage. Several research studies have investigated the use of collagen hydrogels containing different types of collagen, such as type I and type II, in cartilage tissue engineering applications. Kilmer et al. explores the influence of type I collagen on the development and maintenance of tissue and also highlights the potential of collagen-based scaffolds for cartilage tissue engineering and repair25 investigates the effects of the composition and mechanical properties of injectable collagen I/II composite hydrogels on chondrocyte behaviors in cartilage repair. The researchers observed that the microstructure and mechanical property of the hydrogels were relevant to the composition of the composite hydrogels. Hence, discriminating collagen blend gels using P-SHG images is a necessary approach to investigate and quantify the collagen type without the need for staining. Figure 12.. The average peptide pitch angle values of the 0%, 25%, 50%, 75%, and 100% blend hydrogels are 49.01°, 48.47°, 49.46°, 50.16° and 50.89°, respectively. The average anisotropy parameter values are 1.34, 1.41, 1.33, 1.27, and 1.19, respectively. However, the average peptide pitch angle and anisotropy parameter values of the 0% blend hydrogel closely approximate those of the 50% blend hydrogel. Consequently, relying solely on the pixel-wise methods makes it challenging to discern various collagen type I and II blend hydrogels.The study by Yuan et al.Notably, all the polarization images for each collagen blend proportion exhibit distinct differences in intensity as a function of polarization, facilitating the differentiation of collagen fibers. Additionally, an investigation was conducted to assess whether there were discernible differences based on fiber thickness, fiber density, or overall SHG intensity to determine whether the network utilized any of these parameters during its analysis. To ensure unbiased results, all these parameters were kept random during the study.11. However, this method requires fitting polarization SHG pixel-wise information into generic mathematical models, and also reveals inconsistencies for categorizing collagen type I and II blend hydrogel. To address the limitations, the present group sought an automatic and responsive analysis method capable of efficiently differentiating collagen mixtures. Given the capacity of image-based ResNet to function without the need for prior mathematical model fitting and yield precise predictions, the conventional ResNet architecture is modified by incorporating regression and fully connected layers to predict collagen mixtures with varying percentages of collagen type I and type II based on SHG images. Figure x-axis against the standard deviation of each specific concentration.Previously, the present group used pixel-wise analysis with polarization SHG information to discriminate collagen type I and type II in collagen gel assemblyp < 0.25 for 0% type II, p < 0.1 for 25% type II, p < 0.18 for 50% type II, p < 0.15 for 75% type II, and p < 0.15 for 100% type II. In contrast, the model trained with multipolarization images showed lower error bars with p < 0.12 for 0% type II, p < 0.05 for 25% type II, p < 0.1 for 50% type II, p < 0.2 for 75% type II, and p < 0.1 for 100% type II, as shown in Fig. p values for 0% and 25% type II showed an improvement of over twofold. Table Similarly, the 2D multichannel ResNet model trained on the multipolarization SHG images and the predicted vs. actual response for the collagen gel blends is as shown in Fig. p < 0.25 for 0% type II, p < 0.1 for 20% type II, p < 0.11 for 40% type II, p < 0.16 for 60% type II, p < 0.23 for 80% type II, and p < 0.15 for 100% type II in collagen type I hydrogel is observed. Similarly, the 2D multichannel ResNet model trained on the multipolarization SHG images and the predicted versus actual response for the collagen gel blends is shown in the top subplot of Fig. p < 0.11 for 0% type II, p < 0.06 for 20% type II, p < 0.07 for 40% type II, p < 0.15 for 60% type II, p < 0.16 for 80% type II, and p < 0.09 for 100% type II, as shown in the bottom plot of Fig. 28.The proposed model utilizing multipolarization images has shown successful differentiation of the collagen mixture gels at five different proportions. Furthermore, Fig. 29. While the orientation analysis of the collagen network in three dimensions has proven effective in distinguishing healthy tissue from osteoarthritis tissue, the challenge arises from the missing substrate area available within the collagen hydrogel for maintaining collagen cohesion30. ResNet is designed to focus on learning only new, complex information at each layer, making it particularly well-suited for tasks involving large-scale feature extraction. Incorporating polarization images as separate channels in a multichannel ResNet allows the network to capture more diverse and informative features, which can be crucial for tasks such as material classification, object detection, and segmentation31. Considering polarization information allows the model to reduce ambiguity and make more accurate predictions, especially in scenarios where intensity-based features alone are insufficient32. 2D + 1D convolution might be more appropriate where the data can be naturally separated into spatial and spectral components, using a combination of 2D convolutions for spatial information and 1D convolutions for polarization spectral information. This approach is commonly used for processing hyperspectral data, where one has spatial information (2D) and spectral information (1D)33. This is also common in video analysis where each frame (2D) has a temporal sequence. While spatial-spectral CNNs are well-suited for hyperspectral data due to the rich spectral information and high dimensionality, their application to polarization images might not yield the same benefits. Polarization SHG images primarily capture spatial patterns influenced by polarization angles, and the limited angular diversity might not warrant the use of a dedicated 1D processing component. The state-of-the-art 2D ResNet captures the intricate features from the input progressive multipolarization SHG image data and predicts the proportion values as the output. Combining intensity and polarization channels allows the network to fuse complementary information. This can result in a more holistic representation of the scene, enhancing the model's ability to capture complex patterns and structures. To date, although several studies have been conducted to automate the differentiation of collagen fibers in diseases using SHG images, the underlying variation in polarization images has not been well explored. Therefore, this study has the potential to explore the automation of polarization dependent SHG imaging to advance the conventional pixel resolution-based analysis methods. In conclusion, although there is potential in integrating multipolarization SHG images into this ResNet-based regression network, its current ability to differentiate collagen blends may benefit from further optimization.The polarization direction of the incident field determines the polarization direction of the emitted SHG signal and by measuring the polarization profile of the SHG signal at different incident polarization angles, the molecular structure and organization of collagen can be probedThe limitations observed in the pixel-wise analysis emphasize the need for an automated method that can effectively eliminate human intervention and simplify parameter extraction processes, especially when dealing with pure collagen gel types. The proposed multichannel 2D ResNet regression model based on the multipolarization SHG imaging introduces a potential approach for the automated discrimination of collagen blend hydrogel, offering a more efficient and rapid analysis process compared to conventional methods. The utilization of the ResNet model, trained on the multipolarization SHG images at different intervals, showcased a remarkable categorization performance as compared to the model trained on the nonpolarization SHG images. Given time limitations and the primary objective of showcasing the feature extraction potential from multipolarization SHG images, the exploration of combining 3D spatial information with multipolarization SHG images has not been pursued in this study. Overall, the integration of the multipolarization SHG images and the deep learning technique presents a promising avenue for further research in collagen analysis.Fiber samples are prepared in vitro using collagen type I sourced from rat tail  and collagen type II sourced from bovine articular cartilage . Each set of SHG signature comparisons between the two collagen types is conducted with gels polymerized simultaneously to ensure consistent conditions. To ensure sufficient sample size, the experiment is repeated three times for replicates. A working concentration of 1 mg/mL for both collagen types is prepared on ice, and the total gel volume is 0.3 mL, diluted with a phosphate-buffered saline (PBS)  solution. To neutralize the pH of the collagen solution, a sterilized sodium hydroxide (NaOH)  solution is used. The collagen solution is then slowly polymerized in a micro-slide 8-well culture chamber, and maintained at 4 °C for 20 h. The resulting gels, with a thickness of approximately 1.5 mm, are gently released from the bottom of the dish and subsequently fixed in 4% paraformaldehyde  for further analysis. Similarly, all the collagen type I and II blend hydrogels are prepared with 0%, 25%, 50%, 75%, and 100% type II, respectively. Additionally, a few more collagen type I and II blend hydrogels at 0%, 20%, 40%, 60%, 80%, and 100% type II are also prepared to infer the untrained images. The abovementioned procedures of polymerization of collagen fibers are repeated as it is in the case of collagen blend samples.11. Briefly, the system consists of an inverted microscope  with a mode-locked Ti:sapphire laser  serving as the excitation source  and a 20X, 0.7 NA objective lens . Key components of the setup include two galvanometer x and y scanners , a z-axis piezoelectric nano-positioning stage , and a photomultiplier tube (PMT) .The multipolarization SHG imaging setup employed in this study is based on a well-established optical system, as detailed in a prior publicationsee Fig. . The expTo investigate collagen structural data under different polarizations, the setup incorporates two liquid crystal polarization rotators (LPRs) . Linear polarizations are achieved by precisely rotating the first LPR (as LPR1 in Fig. 34. Typically, data analysis for discriminating different collagen types stands on the P-SHG information at the pixel level, whereas DL methods integrate data at the feature level. Here, Fig. A comparative analysis is performed between the two variations of the 2D ResNet model. One variant of 2D ResNet utilizes the nonpolarization SHG images, while the other incorporates multipolarization SHG images presented in a multichannel format. Multi-modality images provide a more comprehensive view of the real world by combining different imaging modalities. This allows for the extraction of various types of information such as thermal radiation differences, texture details, intensity and polarization information, and spatial, spectral, and temporal information


In the era of personalized medicine greatly improved by molecular diagnosis and tailor-made therapies, the survival rate of acute myeloid leukemia (AML) at 5 years remains unfortunately low. Indeed, the high heterogeneity of AML clones with distinct metabolic and molecular profiles allows them to survive the chemotherapy-induced changes, thus leading to resistance, clonal evolution, and relapse. Moreover, leukemic stem cells (LSCs), the quiescent reservoir of residual disease, can persist for a long time and activate the recurrence of disease, supported by significant metabolic differences compared to AML blasts. All these points highlight the relevance to develop combination therapies, including metabolism inhibitors to improve treatment efficacy. In this review, we summarized the metabolic differences in AML blasts and LSCs, the molecular pathways related to mitochondria and metabolism are druggable and targeted in leukemia therapies, with a distinct interest for Venetoclax, which has revolutionized the therapeutic paradigms of several leukemia subtype, unfit for intensive treatment regimens. Acute myeloid leukemia (AML) is an aggressive disease that is still characterized by a dismal prognosis in a significant number of patients: the 5-year overall survival (OS) in the adult population is around 28%, but if patients over 65 are selected, OS is less than 10% .BCL2 associated X), overexpression of MCL1 or other members of the Bcl-2 family, and TP53 mutations) . Th. Th97]. To date, since elevated Bcl-2 expression has been reported in more than 90% of CLL patients, the use of Venetoclax has been extended to relapsed and refractory patients as monotherapy, or in combination with rituximab, obinutuzumab, or ibrutinib . After iThese promising results extended the use of the Venetoclax–HMA regimen as a front-line treatment instead of an induction chemotherapy for naive unfit AML patients. Several studies confirmed the important role of this regimen even in Refractory/Relapsed (R/R) AML patients ,110. IndFLT3-ITD, RAS, or TP53 mutations showed a reduction in sensitivity to Venetoclax-based treatment due to their ability to decrease the LSCs Bcl-2 levels (TP53) [In this scenario, a significant correlation between refractory to Venetoclax and AMLs known for their ability to increase OXPHOS, such as acute monocytic leukemia (AML-M5), has been registered . In addis (TP53) , increass (TP53) ,115, or s (TP53) . Howevers (TP53) .Due to its relevance, the biological basis for Venetoclax resistance has been extensively studied. To date we can identify the main mechanisms involved : occurreFinally, identifying biomarkers  after Venetoclax treatment) that can predict which patients are more eligible to develop resistance to Venetoclax is an active area of research that may help to guide treatment decisions .In conclusions, it is now clear that leukemic stem cells have a peculiar metabolism that can be exploited for inducing a selective death of tumor cells. A better knowledge of the mitochondrial metabolism of the leukemic cells, as well as of the drug-resistance mechanisms that aim to act against these metabolic pathways, could represent the basis for an increasingly personalized and effective therapy.


Acute myeloid leukemia (AML) is a dangerous cancer of the blood. In recent years, a series of drugs was approved to specifically target misdirected processes in the cancerous cells. These so-called “small molecules” substantially improved therapeutic outcomes, but eventually leukemia returns in most patients. In this review, we summarize the current state of knowledge regarding the mechanisms that lead to failure of the most frequently used new therapies and introduce potential strategies to overcome the mechanisms associated with disease recurrence.In recent years, great progress has been made in the therapy of AML by targeting cellular processes associated with specific molecular features of the disease. Various small molecules inhibiting FLT3, IDH1/IDH2, and BCL2 have already gained approval from the respective authorities and are essential parts of personalized therapeutic regimens in modern therapy of AML. Unfortunately, primary and secondary resistance to these inhibitors is a frequent problem. Here, we comprehensively review the current state of knowledge regarding molecular processes involved in primary and secondary resistance to these agents, covering both genetic and nongenetic mechanisms. In addition, we introduce concepts and strategies for how these resistance mechanisms might be overcome.  AML is an aggressive blood cancer characterized by uncontrolled proliferation of malignant hematopoietic stem and progenitor cells. Comprehensive genomic studies have revealed the genetic complexity of this disease, leading to refined classification systems and risk stratification ,2,3,4. For a long time, therapy of AML has been purely chemotherapy-based, with cytarabine and anthracyclines being the standard of care in most first-line settings ,6. HowevIn this review, we focus on the current knowledge of genetic and nongenetic mechanisms of resistance of the clinically most relevant, authority-approved small molecules in the context AML. These comprise various FLT3 inhibitors, inhibitors of IDH1/IDH2, and a BCL2 inhibitor. To this end, PUBMED was searched for the terms listed in the keywords section and names of individual inhibitors , and the literature was critically reviewed. The review presented here focuses on articles providing clinical or mechanistic reports on resistance to small molecules in AML therapy. FLT3 gene located on chromosome 13q12. It is primarily expressed in hematopoietic progenitor cells [FMS-like tyrosine kinase 3 (FLT3) is a protein encoded by the or cells  and is oor cells . FLT3 coor cells . This acFLT3 mutations [FLT3 mutations often exhibit high leukocyte and blast counts in both peripheral blood and bone marrow [Roughly one-third of patients with AML have activating utations . In apprutations . Patiente marrow .FLT3-ITD mutations in the intermediate risk group. This reclassification is primarily due to the fact that FLT3 mutations have become a “druggable” target with the development of FLT3 inhibitors, which nicely demonstrates that targeted therapy can impact disease outcome and the prognostic value of genomic aberrations [In contrast to the previous version of the European LeukemiaNet (ELN) risk classification of AML, the most recent version categorizes all rrations . FLT3-ITD-positive AML in the SORMAIN study [FLT3-mutated AML within the large, randomized phase-III RATIFY trial. In recent years, several small-molecule tyrosine kinase inhibitors (TKIs) targeting the ATP-binding site of the FLT3 kinase or adjacent structures have been developed ,15. FirsIN study . In addiIN study ,19. WhilIN study , and redIN study  in FLT3-FLT3-ITD mutations compared to standard salvage chemotherapy in the QUANTUM-R trial [FLT3-ITD-mutated AML within the QUANTUM-First study [FLT3-mutated AML. It gained approval from the FDA and EMA based on the data from the ADMIRAL study [Second-generation FLT3 inhibitors more specifically bind to FLT3 than first-generation FLT3 inhibitors and, hence, have shown improved efficacy. They include quizartinib, gilteritinib, and crenolanib . Quizart-R trial . Lately,st study . GilteriAL study . FLT3-TKD-mutated disease. Midostaurin, gilteritinib, and crenolanib belong to the group of type-1 inhibitors, while sorafenib and quizartinib are classified as type-2 inhibitors [FLT3 inhibitors can also be classified into type-1 and type-2 inhibitors. While type-1 inhibitors bind to the active conformation of FLT3, type-2 inhibitors bind to its inactive conformation. Pharmacologically, type-1 inhibitors can target both ITD and TKD mutations, while type-2 inhibitors do not effectively inhibit hibitors ,24. Despite promising results, response durations in patients treated with FLT3 inhibitors remain relatively short when used as monotherapy in the r/r setting (4–14 months) ,23. TherFLT3 mutations has been identified as a mechanism of secondary resistance to treatment with FLT3 inhibitors. FLT3 F691L, a well-known gatekeeper mutation within the active site of the TKD, confers resistance to all clinically used FLT3 inhibitors [FLT3 N676K in TKD, which was shown to confer resistance to midostaurin [FLT3 D835 (D835F/V/Y) or FLT3 Y842C/H, only confer resistance to type-2 FLT3 inhibitors like quizartinib [FLT3 mutations is also considered one of the main factors contributing to the drop in composite CR (CRc) rates during sequential exposure to FLT3 inhibitors [FLT3-ITD-mutated AML demonstrated that secondary FLT3-ITD mutations occurred in only 11% of patients at the time of r/r disease. Instead, the authors observed an outgrowth of clones that lost the FLT3-ITD mutation and clones harboring signaling pathway mutations downstream of FLT3 as the most prevalent mechanisms of resistance in their patient cohort [On-target mutations A: The achibitors ,26,27,28ostaurin , and K42ostaurin . Mutatiozartinib . The emehibitors . HoweverRAS/MAPK pathway genes (13%), WT1 (7%), and TP53 (7%). Mutations in genes of the RAS/MAPK pathway were commonly observed as mechanisms of resistance to type-1 FLT3 inhibitors (29%). Nonresponders to FLT3 inhibition had a higher variant allele frequency (VAF) of RAS mutations  as identified NGS, likewise indicating a role of the RAS pathway in this context [WT1, NRAS, KRAS, and IDH1. In addition, the authors observed acquired mutations in genes associated with chromatin cohesin/splicing  upon resistance development in these patients, indicating a role for associated processes in the development of resistance [NRAS, PTPN11, ABL1, BCORL1, CEBPA, WT1, and IDH1 emerged under treatment with crenolanib in another study, leading to resistance, as shown by WES [Activation of alternative signaling pathways by off-target mutations B: Variou context . On a sisistance . Similarn by WES . In a stn by WES .Over-expression of antiapoptotic proteins C: The ovRole of microenvironment/stem cell niche D: The boSeveral approaches exist to overcome resistance to FLT3 inhibition. These include the development of next-generation inhibitors, combination therapies with cytotoxic chemotherapy or other targeted agents, targeting the microenvironment, and utilizing dual-targeted inhibitors.FLT3-ITD mutations. Of note, the predicted binding sites of sitravatinib do not include the F691L residue [A series of next-generation FLT3 inhibitors are currently at different stages of preclinical and clinical investigation. Among these, sitravatinib was more effective than gilteritinib in xenograft models derived from patient blasts carrying  residue . The cov residue . It also residue . FLT3 molecular response rates, even in patients with prior FLT3 inhibitor treatment [FLT3 wild-type high-risk AML. Mechanistically, the combination led to downregulation of antiapoptotic protein myeloid leukemia 1 (MCL1) via altered signaling of the involved ERK pathway [Another strategy to overcome resistance to FLT3 inhibition is the addition of the BCL2 inhibitor venetoclax to induce apoptosis. A multitude of studies have demonstrated the synergistic nature of this approach in the preclinical setting ,52,53,54reatment . Interes pathway . Combina pathway ,58,59, a pathway  or CXCR4 pathway  inhibiti pathway . IDH1 and R140 and R172 in IDH2 [The isocytrate dehydrogenase (IDH) enzymes catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG). Recurrent mutations in the iso-enzymes IDH1 and IDH2 occur in approximately 20% of de novo AML . These m in IDH2 ,64,65. T in IDH2 . 2-HG co in IDH2 , includi in IDH2 ,69. Inhi in IDH2 ,71.IDH1-mutated AML can be effectively treated with the orally available IDH1 inhibitor ivosidenib. Effectiveness as monotherapy in newly diagnosed AML with IDH1 mutation was demonstrated in patients ineligible for intensive chemotherapy [IDH1-mutated AML was likewise shown [IDH1-mutated AML ineligible for intensive chemotherapy in the phase-III AGILE trial. Here, the combination of ivosidenib with azacytidine led to significantly improved EFS and OS [IDH1 mutation in r/r AML or elderly patients ≥75 years or not suitable for intensive induction chemotherapy in 2018 and 2019, respectively. In 2022, ivosidenib was approved as a first-line therapy in combination with azacytidine for patients with IDH1 R132 mutation not eligible for intensive induction chemotherapy. EMA approval for this combination therapy followed in May 2023. otherapy . Effectise shown . EncouraS and OS . Based oOlutasidenib is another orally available FDA-approved IDH1 inhibitor. In r/r patients with AML, olutasidenib led to overall response rates of almost 50% and a median duration of response of 25.9 months in those patients achieving CR/CRh  , which iIDH2 that was granted FDA approval for r/r AML in 2017. In a pivotal phase-I/II study, enasidenib led to overall response rates of approximately 40% in patients with IDH2-mutated AML. Of note, while median OS was 9.3 months for the total cohort, patients attaining CR (19.3%) had a median OS of 19.7 months [IDH2 mutation, overall response rates of more than 30% were achieved [IDH2 mutation, the combination of enasidenib and azacytidine led to a significantly improved overall response when compared to azacytidine alone [Enasidenib is a first-in-class orally available inhibitor of mutant 7 months . In a stachieved . Likewisne alone . Mechanisms of primary or secondary resistance to IDH inhibitors are manifold and comprise genetic, as well as nongenetic, mechanisms. IDH1 clone size and achievement of CR following ivosidenib monotherapy [IDH2 clone size and response to single-agent enasidenib therapy [VAF/clone size: It is conceivable that the clone size can affect response and resistance patterns to targeted therapy. However, according to a study by Choe et al., there was no correlation between mutant otherapy . Similar therapy . IDH1- and IDH2-mutated AML treated with ivosidenib and enasidenib, respectively. In a study by Choe et al. investigating mechanisms of resistance in 179 patients treated with ivosidenib, 20 second-site mutations in IDH1 were detected at relapse or progression that either affected the binding pocket of ivosidenib or its cofactor NADPH or that led to hypothesized structural changes, preventing the interaction between ivosidenib and IDH1 [IDH2 mutant AML. In specific, Intlekofer et al. describe two cases with newly acquired mutations on the second IDH2 allele, which led to restoration of 2-HG production and AML relapse [Escape mutations in IDH enzymes/second site mutations A: Escapeand IDH1 . Likewis relapse .IDH2 mutations in the context of IDH1 inhibition and vice versa. This mechanism was described by Harding et al. in a series of two patients with AML and IDH1 mutation receiving ivosidenib. After initial response to the therapy, both patients suffered from relapse that could be attributed to the outgrowth of IDH2 R140Q-mutated clones [IDH2-mutated AML treated with enasidenib that developed a de novo IDH1 mutation at relapse [Isoform switching B: Anothed clones . The samd clones . Likewis relapse . RAS genes [FLT3 [IDH2 mutant patient who had received enasidenib, leukemic clones harboring NRAS and KRAS mutations arose independently from the IDH2 mutant clone, highlighting clonal selection processes [RUNX1 and CEBPA have been found in patients with both primary or secondary treatment failure of IDH inhibition [IDH2-mutated AML [Co-occurring mutations C: Co-occAS genes ,81,85 anes [FLT3 ,84,85,86hibition ,84. ThisIDH2 R172 mutation showed a significantly stronger reduction in 2-HG levels than nonresponders. However, in patients with IDH2 R140 mutation, 2-HG levels were effectively suppressed, irrespective of response category [Levels of 2-HG: Contradictory data exist for the question of whether the suppression levels of 2-HG correlate with response or resistance to IDH inhibition. In a study by Stein et al., patients with category . On the category . While scategory . Leukemia stemness D: In theAlthough responses to IDH inhibition are often long-lasting, relapses eventually occur. As outlined above, clonal heterogeneity and outgrowth of resistant clones seem to be major sources of resistance. Mutations that confer resistance often affect pathways not directly related to IDH inhibition , highlighting the necessity of hitting multiple targets using combination therapies. With respect to IDH inhibitors, multidrug combination regimens with HMAs and BCL-2 inhibitors yield promising results and are detailed in BCL2 and the related proteins BCLXL and MCL1 are antiapoptotic proteins that prevent apoptosis by stabilizing the outer mitochondrial membrane, thus circumventing permeabilization and cytochrome c release . BCL2 isVenetoclax is an orally available BH3 mimetic that inhibits BCL2 and, hence, induces apoptosis via release of the proapoptotic proteins BAK and BAX. In patients with AML, it has limited efficacy when used as a monotherapy in r/r patients . HoweverThe mutational pattern of AML plays an important role in response and resistance to venetoclax and can be used to predict response to therapy and relapse risk. A relevant number of mutations are associated with favorable outcomes in this setting and are, therefore, briefly introduced here as well.IDH1 and IDH2 is associated with a favorable response pattern in monotherapy [IDH1/IDH2-mutated cells suppressed cytochrome c oxidase (COX) and, thus, lowered the threshold of mitochondria to initiate apoptosis via BCL2 inhibition. Therefore, IDH1/IDH2 mutations and BCL2 inhibition are an example of synthetic lethality [TET2 are likewise associated with favorable response to venetoclax-based therapeutic regimens [NPM1 mutations have been associated with favorable outcome parameters such as higher CR rates, higher blast reductions, or increased OS [SRSF2 and ZRSR2 have been reported to be associated with beneficial outcomes in studies involving venetoclax in AML treatment [Mutations associated with response A: A multotherapy ,94, as wotherapy ,96, low-otherapy , or inteotherapy . An in vethality . Due to regimens . Likewiseased OS ,101,102.eased OS . In addiFLT3 and TP53 has been associated with inferior treatment outcomes [FLT3-ITD-bearing subclones appeared early in treatment, with clinical relapse from one to six months after therapy initiation. In the same study, relapse was driven by emergence of newly diagnosed FLT3-ITD mutations in two patients. Interestingly, resistance to venetoclax was also mediated by polyclonal resistance mechanisms, as demonstrated through single-cell sequencing. Here, resistance was due to a series of independent clones that harbored individual mutations in FLT3 and NRAS. In the same study, clonal selection of mutations in TP53 contributed to one-third of relapses, and clonal outgrowth of TP53 was observed in all previously mutated cases. In line with these findings, primary resistance was associated with mutations in activating kinases , TP53, and RUNX1 [FLT3-ITD or PTPN11 mutation at baseline. Likewise, these mutations were associated with primary resistance to venetoclax in this study [Mutations associated with resistance B: In mosoutcomes ,101,102.outcomes ,97. In ais study . Monocytic differentiation C: IntereEnergy metabolism D: In addThere are various approaches to overcoming resistance to BCL2 inhibition, comprising various stages of preclinical and clinical testing. In the preclinical setting, inhibitors of BCLXL or MCL1 showed strong synergistic killing when combined with venetoclax in resistant AML cell line models with elevated levels of these alternative proapoptotic proteins . S64315,IDH-mutated AML favorably responds to venetoclax-based therapy [FLT3-mutated patients with AML with a 2-year OS of 80% [FLT3-mutated patients with AML, irrespective of prior exposure to FLT3 inhibitors [On the clinical side, the combination of venetoclax with other specific targeted therapies is currently being tested with the aim to achieve longer remissions and to prevent secondary resistance. As discussed,  therapy ,91. To f therapy . Other cS of 80% . Similarhibitors . Pharmacological inhibition of FLT3, IDH, and BCL2 are the most advanced therapeutic options involving small-molecule inhibitors in AML. However, new avenues are being explored for the treatment of AML, such as TP53 reactivation, menin inhibition, or E-selectin inhibition. In addition, glasdegib is another approved small molecule targeting hedgehog signaling. Likewise, HMAs should also be mentioned in the context of this review. DNMT3A mutations negatively impacted OS in patients with secondary AML within the BRIGHT AML 1003 study [Based on the BRIGHT AML 1003 study, the hedgehog-signaling inhibitor glasdegib has been approved by the FDA and EMA in combination with low-dose cytarabine for patients ineligible for intensive chemotherapy. Resistance to glasdegib is mechanistically not well understood, but the presence of 03 study ,116.IDH [DNMT3A [TET2 [RUNX1 and SRSF2 were associated with resistance to HMA therapy [Azacytidine and decitabine are the HMAs most frequently used, either alone or in combination with various other antileukemic agents. They cause hypomethylation of DNA, and mutations in epigenetic modifiers such as IDH , DNMT3A  [DNMT3A , and TET3A [TET2  have bee therapy . Likewis therapy . Interes therapy . However therapy . TP53 is mutated in 5–15% of AML cases and is associated with a poor prognosis in AML and MDS [ and MDS  and was  and MDS . Data onNPM1 [MEN1 affecting the revumenib–menin interface could confer acquired resistance to menin inhibition. These findings were also validated in xenograft models. Of note, the study was the first to demonstrate that chromatin-targeting therapeutic drugs could impose sufficient selection pressure to drive the evolution of escape mutants [Menin is a scaffold protein that is essential for AML with KMT2A fusion proteins or mutated NPM1 . Menin iNPM1 ,128. Acc mutants . Another interesting target in AML treatment is E-selectin, as leukemic (stem) cells that bind to E-selectin in vascular niches become more resistant to chemotherapy . This prIn less than a decade, small molecules have revolutionized the therapeutic landscape of AML, greatly enlarging the treatment portfolio and improving outcomes. Except for the BCL2 inhibitor venetoclax, most small molecules target molecular lesions that occur only in subsets of AML , making them a substantial part in the therapeutic concept of precision medicine. Promising new agents targeting different pathogenetic processes in leukemic blasts are under development, leaving us with the hope that we might be able to offer targeted therapies to patients with AML subtypes that cannot yet benefit from this therapeutic concept. However, euphoria over targeted treatments is often disrupted by primary resistance or relapses that eventually occur in almost all cases. As discussed in detail in this review, leukemic cells possess a multitude of mechanisms to overcome pharmacologic inhibition. These mechanisms comprise various kinds of molecular processes, from nucleus to cell-to-cell interactions, and we expect that many more will be discovered in the future. Irrespective of the specific nature of an escape mechanism, highly effective clonal selection processes and associated rapid evolution of AML leave physicians and researchers with the challenge of how to durably eliminate this dynamic and adaptable disease.Combining various small molecules to simultaneously target both a specific lesion and important escape mechanisms appears to be a promising approach  and leaves us with a growing number of therapeutic options. Among the multitude of escape mechanisms, clonal outgrowth of RAS/RTK pathway mutations seems to be a frequent pattern of resistance to targeted therapies in AML, as well as in other myeloid malignancies ,134. ComIn summary, the small molecules currently available provide a first step in the direction of precision medicine for patients with AML. In the future, sophisticated combination regimens and comprehensive molecular testing will hopefully help us to refine personalization strategies for leukemia treatment and, hence, achieve more and longer-lasting responses.


The impact of genes involved in drug transport and metabolism in regard to the lack of response of acute myeloid leukemia (AML) cells to induction chemotherapy using cytarabine and idarubicin has been investigated in blast cells collected at diagnosis. The aim of this study was to evaluate the usefulness of measuring their expression in order to predict the response to induction chemotherapy. In AML patients with a lower response, the elevated expression of uptake and export transporters and enzymes was found. Additionally, AML cell lines more sensitive to cytarabine showed altered levels of these genes. In conclusion, the poor response of AML patients to chemotherapy can be associated with the increased expression of inactivating enzymes, likely resulting in a reduced intracellular concentration of the active cytarabine metabolite in their blasts.Despite its often low efficacy and high toxicity, the standard treatment for acute myeloid leukemia (AML) is induction chemotherapy with cytarabine and idarubicin. Here, we have investigated the role of transporters and drug-metabolizing enzymes in this poor outcome. The expression levels (RT-qPCR) of potentially responsible genes in blasts collected at diagnosis were related to the subsequent response to two-cycle induction chemotherapy. The high expression of uptake carriers (ENT2), export ATP-binding cassette (ABC) pumps (MDR1), and enzymes  in the blasts was associated with a lower response. Moreover, the sensitivity to cytarabine in AML cell lines was associated with ENT2 expression, whereas the expression of ABC pumps and enzymes was reduced. No ability of any AML cell line to export idarubicin through the ABC pumps, MDR1 and MRP, was found. The exposure of AML cells to cytarabine or idarubicin upregulated the detoxifying enzymes (5-NT and DCK). In AML patients, 5-NT and DCK expression was associated with the lack of response to induction chemotherapy (high sensitivity and specificity). In conclusion, in the blasts of AML patients, the reduction of the intracellular concentration of the active metabolite of cytarabine, mainly due to the increased expression of inactivating enzymes, can determine the response to induction chemotherapy. One of the main problems in the management of patients with acute myeloid leukemia (AML) is that a significant number of cases indicate a poor response to available chemotherapy, or relapse due to the development of chemoresistance, which leads to very unsatisfactory outcomes . The admCytarabine is taken up across the plasma membrane of leukemia cells by members of the equilibrative (ENT) and concentrative (CNT) families of nucleoside transporters . AdditioThere is some controversy regarding the mechanism accounting for idarubicin (4-demethoxy-daunorubicin) hydrochloride uptake. In aqueous solution at physiological pH, this compound has a net positive charge. However, it has been proposed that due to its marked lipophilicity, idarubicin can cross the plasma membrane by means of a flip-flop mechanism . In contOur study aimed to investigate the role of transporters involved in the uptake and efflux of cytarabine and idarubicin and the presence of drug-metabolizing enzymes in leukemia cells (hereafter referred to as blasts) in the response of AML patients to induction chemotherapy and also to evaluate whether their expression levels in blasts collected at diagnosis could be useful to predict the failure of this treatment.2 for 7 days), plus intravenous idarubicin (12 mg/m2 from days one to three), and an equal second cycle, or one consisting of a cytarabine and/or idarubicin dose adjustment, if cryopreserved samples at diagnosis were available. The research protocol was approved by the Ethics Committees for Clinical Research of Salamanca University Hospital and Gregorio Marañón University Hospital, and informed written consent was obtained from each patient for the samples to be used for biomedical research.Blasts were obtained from the bone marrow samples of 67 patients from the Salamanca University Hospital and the Gregorio Marañón General University Hospital with newly diagnosed AML between 2008 and 2016. Patients were eligible if they received standard front-line induction therapy 7 + 3, consisting of a first cycle of cytarabine . Patients were classified as responders (R) if they achieved CR or CRi after two cycles. Patients dying during induction and before response assessment were considered as non-evaluable. The remaining patients were classified as non-responders (NR).Clinical and laboratory test values were collected from electronic and paper medical records. The response to induction chemotherapy was assessed according to the revised Cheson criteria . Complet2. All cell lines were routinely tested by PCR to ensure that they were free of Mycoplasma contamination .Cell lines MOLM-13, HL-60, HEL, derived from AML, and K-562, derived from chronic myeloid leukemia in the acute phase resembling AML, were purchased from the ATCC or DSMZ  cell line repositories and were cultured in RPMI medium supplemented with 10% heat-inactivated fetal calf serum and penicillin-streptomycin and were incubated at 37 °C in a humidified atmosphere with 5% CO2 for 4 h. Purple formazan crystals were solubilized overnight in 100 µL of 10% SDS in HCl 0.01 M, and subsequently, the absorbance was measured in a microplate reader at 595 nm. The drug concentration required to reduce cell viability by 50% (LC50) was calculated from the dose–response curves.Cell viability was evaluated with the MTT-formazan test using thiazolyl blue tetrazolium bromide (MTT) . Briefly, the cell suspensions were diluted with media containing graded concentrations of cytarabine or idarubicin, alone or in combination with inhibitors. Next, the cell suspensions were seeded in 96-well microtiter plates . All drug concentrations and controls (untreated cells) were assayed in triplicate in each culture. After 72 h, MTT was added to each well, and the microplates were maintained at 37 °C with 5% CO−ΔΔCt method was applied to analyze the relative expression of each gene. The results of mRNA abundance for target genes in each sample were normalized compared to GAPDH mRNA abundance. For each cell line, the analysis was performed in cDNA obtained from three independent cell cultures, and the quantification was performed in duplicate. In each human sample, all analyses were carried out in triplicate.Total RNA was extracted from mononuclear cells that were isolated from bone marrow samples using Ficoll-Paque Plus , or from cell lines using the illustra RNAspin Mini RNA Isolation Kit  . Reverse2PO4, 0.8 mM MgSO4, 1.8 mM CaCl2, 11 mM glucose, and 50 mM buffer HEPES/Tris, pH 7.40) containing 50 nM DiOC2(3), 0.1 µM calcein AM, 1 µM carboxyfluorescein, or 25 μM mitoxantrone at 37 °C for 30 min (loading period). Then, the cells were diluted to 1:10 with a substrate-free TM in the presence or absence of a typical inhibitor of each pump, i.e., verapamil (MDR1 inhibitor), probenecid (MRP1-5 inhibitor), diclofenac (MRP3-5 inhibitor), or fumitremorgin C (BCRP inhibitor)   and incuThe contribution of ABC pumps to idarubicin and doxorubicin efflux was also investigated in HEL and MOL-13 cells using flow cytometry. The cells were incubated with 5 µM idarubicin or 25 µM doxorubicin at 37 °C for 30 min (loading period) and then diluted to 1:10 with a substrate-free TM, with or without the selected ABC inhibitors, and incubated at 37 °C for 30 min. After adding 900 μL of ice-cold TM, the fluorescence of these anthracyclines was determined.t-tests or Kruskal–Wallis H-tests were used to compare groups (paired or unpaired). Categorical variables were subjected to the Chi-square test. Unadjusted p-values below 0.05 were regarded as statistically significant. Analysis of the receiver operating characteristic (ROC) curve was completed to evaluate the discrimination ability of the variables. Analysis was performed in terms of area under the receiver-operating characteristic curve (AUC) using R software v4.0.3 . Multivariate analysis, including principal component analysis (PCA), was also performed using this software.Either means and standard error or median and range were used to describe continuous variables. Two-tailed independent Student’s A total of 75 bone marrow samples were obtained from AML patients at two Spanish hospitals. A total of eight samples were excluded from the study due to insufficient RNA being extracted from the isolated blasts or due to early death that precluded the analysis of the association with the treatment response. The main clinical characteristics of the patients recruited at diagnosis are summarized in According to the mutational status of nucleophosmin 1 (NPM1) and the fms-related tyrosine kinase 3-internal tandem duplication (FLT3-ITD) of ELN 2017 risk stratification , and conThe potential clustering of the different groups of patients according to the expression of the selected genes was assessed by multivariate data analysis and unsupervised PCA. No differences were found in gene expression profiles considering the hospital of origin , leucocyA random distribution of patients that achieved CR or CRi after one cycle (R1) or two cycles (R2) of induction therapy, or with no response after the two cycles of treatment (NR), was found . Since tThe mRNA levels of transport proteins involved in cytarabine uptake determined in AML blasts obtained at diagnosis are shown in Regarding export pumps, the order of mRNA abundance was MRP1 > MRP5 > MRP4 > MDR1 > BCRP > MRP8. Higher levels of MDR1 were found in NR than in R patients A, and noThe expression of DCK was lower in the blasts of R patients A, which We investigated whether there was any association between the expression of the genes of interest. High levels of ENT2, MDR1, DCK, 5-NT, and CDA mRNA in the blasts at diagnosis were associated with a lower response to induction therapy. Two genes, namely 5-NT and DCK, showed higher values of sensitivity and acceptable specificity, permitting the correct detection of ≥75% of the AML patients who will respond to induction chemotherapy.We then tested whether the combination of two or more of these genes associated with chemoresistance increased the ability to predict the lack of response to treatment. The analysis revealed that when considering the upregulation of two of these genes, the sensitivity and specificity results were 58.3% and 85.2%, respectively. If enhanced expression of three genes was considered as the selection criterion, the values showed 50% sensitivity and 96.3% specificity. Finally, if the upregulation of four genes was used for the analysis, the specificity was optimal (100%), but the sensitivity dropped to 16.7%.The cytostatic effects of cytarabine and idarubicin were determined in a panel of three AML human cell lines; HL-60, MOLM-13, and HEL, as well as one derived from chronic myeloid leukemia in the acute phase, resembling AML (K-562).50). The sensitivity to cytarabine was higher for HL-60 > HEL ≈ MOLM-13 >> K-562, and the sensitivity to idarubicin was higher for MOLM-13 > HL-60 > HEL >> K-562.The dose–response curves of cytarabine and idarubicin are shown in CNT3 levels were low in HL-60 > MOLM-13 ≈ HEL cells and undetectable in K-562 cells. The expression of ENT1 was higher in MOLM-13 > HL-60 > K-562 > HEL. The expression of ENT2 was high in all the cell lines, especially in MOLM-13 > HL-60 ≈ HEL > K-562.The basal mRNA levels of nucleoside equilibrative transporters ENT1-2 and concentrative transporters CNT1-3 were determined . The CNTRegarding the basal mRNA levels of export pumps , MDR1 waRegarding the basal expression of cytarabine metabolism enzymes , the expThe exposure to drugs induced changes in the expression of genes associated with chemoresistance . IncubatAs previously described , the funGiven that there is controversy regarding the ability of MDR1 to transport idarubicin, we selected HEL and MOLM-13 cells, with high and very low levels of this efflux pump, respectively, to compare their ability to export this drug. As shown in In both in HEL and MOLM-13 cells, after a loading period with idarubicin, the cell content decreased progressively over 30 min after removing the drug from the incubation medium C. Drug eWe also investigated the activity of MRPs involved in the efflux of idarubicin. As shown in Finally, we investigated whether ABC pump inhibitors can sensitize AML cells to cytarabine and idarubicin as an indirect method of determining whether these transporters are involved in the efflux of these drugs. HEL and MOLM-13 cells were exposed to increasing concentrations of the MDR1 inhibitor verapamil A, the MRThe identification of prognostic biomarkers in AML patients is required to help clinicians to choose between conventional and investigational therapies  and to aAmong the mechanisms responsible for resistance to the drugs administered in induction therapy for AML are changes in the expression of some proteins responsible for the uptake and efflux of cytarabine and idarubicin, as well as the enzymes that activate and inactivate these drugs, as previously described ,29. TimeThe processing of the drugs in the liver may affect their pharmacokinetics and hence, their efficacy and toxicity, but the expression levels of these genes in tumor cells can also determine the amount of active intracellular agents and can play an important role in intrinsic and acquired resistance . In thisAlthough there is evidence supporting that MRP4 and MRP5 can transport cytarabine , no relaThere is some controversy regarding the role of MDR1 in AML chemoresistance. Although various studies have failed to detect any association between MDR1 expression and the response of AML patients to treatment with induction therapy ,18, otheResults in AML cell lines showed that changes in transporters can only partially explain drug resistance in these cellular models. HL-60 cells were the most sensitive to cytarabine, which could be associated with high ENT2 expression, as well as low levels of efflux pumps (MRP4 and MRP5) and inactivating enzymes (5-NT and CDA). MOLM-13 cells were the most sensitive to idarubicin, but MRP1 levels were similar to those found in K-562 cells, which are the most resistant to this drug. Nevertheless, low ENT2 expression and high levels of MRP1 and MRP5 alone cannot justify the high resistance to both drugs. Additionally, MDR1 and MRPs inhibitors did not sensitize AML cells to idarubicin or cytarabine.The balance between the activity of the three enzymes involved in nucleoside metabolism assayed here is one of the crucial factors determining the concentration of the active metabolite of cytarabine within AML cells ,9. The lIn blasts from AML patients, the intracellular concentration of the active metabolite of cytarabine is expected to be reduced by the upregulation of the inactivating enzymes 5-NT and CDA. Moreover, the high expression of drug transporters (ENT2 and MDR1) and metabolizing enzymes  in these cells is associated with a worse response to induction therapy. Larger scale validation studies are needed to confirm the clinical diagnostic value of these biomarkers.


While multiple families of Ca2+-permeable channels at the plasma membrane have been uncovered, the counterparts responsible for Ca2+ release from intracellular stores remain poorly understood. In a groundbreaking paper published recently by Cell, the authors reported that WeiTsing, an Arabidopsis endoplasmic reticulum (ER)-resident protein that was specifically expressed in the pericycle upon Plasmodiophora brassicae (Pb) infection, could form resistosome-like Ca2+-conducting channel and protect the stele of Brassica crops from Pb colonization. As the channel activity of WeiTsing was indispensable for its immune function, the findings highlight a previously underappreciated role of Ca2+ release from intracellular repertoire in promoting plant disease resistance.Plants employ pattern- and effector-triggered immunity (PTI and ETI) to synergistically defend invading pathogens and insect herbivores. Both PTI and ETI can induce cytosolic Ca Meanwhile, locally occurring PTI or ETI can induce the production of secondary mobile molecules that travel to distal tissues to trigger systemic acquired resistance (SAR) against subsequent attacks.Lacking an adaptive immune system, plants solely rely on the innate immune system to counteract pathogen infection and herbivore infestation. Plant innate immunity can be conceptually categorized into pattern-triggered immunity (PTI) and effector-triggered immunity (ETI)  to avoid forming insoluble and cytotoxic Ca3(PO4)2 with abundant phosphate ions, while free Ca2+ at much higher concentrations is sequestered in the apoplast (~ 10 mM) and intracellular organelles, such as vacuoles (up to 5 mM) and endoplasmic reticulum   are subsequently deciphered by various types of Ca2+ decoders, such as calmodulins (CaMs), Ca2+-dependent protein kinases (CDPKs) or metacaspases , ANNEXINs (ANNs), MILDEW RESISTANCE LOCUS Os (MLOs), and the ion channels formed by coiled-coil (CC) or helper NLR complexes , which can form a dimeric channel on the tonoplast   in this region, among which only C6 could lead to an autoimmune phenotype in Col-0 when expressed by a 2-kb native promoter, whereas C6 knockout in Est-1 caused Pb susceptibility. Of particular note, when C6 (later renamed WeiTsing) was expressed by a 3.6-kb native promoter (p3.6k) in Col-0, the transgenic plants appeared to grow normally while retaining Pb resistance. Transgenic introduction of p3.6k-WeiTsing into the oilseed rape (Brassica napus) also conferred broad-spectrum Pb resistance without growth penalty. These results suggest that WeiTsing is the clubroot resistance gene and its activity is tightly modulated by transcriptional regulation. Consistently, the authors found that WeiTsing was expressed at low levels in un-inoculated Est-1 roots, but was massively induced by inoculation with all 16 Pb isolates. Interestingly, the promoter of WeiTsing in the Ler accession was defected. Accordingly, WeiTsing in Ler could not be induced by Pb infection to confer resistance, whereas transgenic introduction of the p3.6k promoter from Est-1 in combination with the WeiTsing coding sequence from Ler could establish Pb resistance in Col-0, suggesting that the p3.6k promoter is key for Pb resistance. Noteworthily, a preprint paper also reported the same gene (named RPB1) as a Pb resistance gene , suggesting that WeiTsing specifically functions in the pericycle to protect the stele from Pb colonization.Next, Wang and colleagues examined the tissue specificity and inducibility of the WeiTsing mediate Pb resistance in the pericycle? To answer this question, the authors looked into WeiTsing-dependent transcriptional changes in Arabidopsis and oilseed rape upon Pb inoculation and found that the Pb-induced WeiTsing expression led to transcriptional activation of many defense-related genes. Moreover, the estradiol-induced WeiTsing expression was sufficient to trigger immune responses in the absence of Pb infection. Notably, WeiTsing was localized to the ER and could form oligomers in both co-immunoprecipitation and gel infiltration assays. The oligomeric structure of WeiTsing was subsequently solved by cryo-electron microscopy, which exhibited a symmetrical pentameric architecture with a central pore, reminiscent of the CC-NLR-containing resistosome. By using multiple electrophysiological approaches, the authors verified the pentameric WeiTsing complex as a cation-selective channel permeable to Ca2+. Taking advantage of the Ca2+ sensor GCaMP6m, they provided in planta evidence that WeiTsing is a Ca2+-permeable channel. Structure-guided mutagenesis further indicated that WeiTsing-mediated immune responses require its channel activity.Then, how does Pb stimulates the expression and activity of an ER-resident Ca2+-conducting channel in pericycle cells, leading to fortified pericycle immunity to safeguard the stele. This study not only greatly improves our understanding about the intracellular Ca2+ channels controlling organellar Ca2+ release but also opens up a new avenue for genetic engineering of clubroot resistance in Brassica crops. Future studies are needed to determine how Pb induces WeiTsing expression in Est-1and how WeiTsing coordinates with PM-localized Ca2+ channels to regulate Pb-induced Ca2+ signature. The mechanism for reprograming the expression of defense-related genes by WeiTsing-mediated Ca2+ signaling in the pericycle upon Pb infection also remains to be characterized. Furthermore, because many soil-borne pathogens can breach the stele to cause diseases, it is tempting to speculate that WeiTsing also positively regulates the pericycle immunity against some of those pathogens.In summary, Wang et al.  elegantl


Wisteria Floribunda agglutinin were consistently smaller than the DLZ/CA2 borders by ~100 μ on the CA1 or CA3 sides respectively. In addition, these patterns are dependent on position along the dorsal–ventral hippocampal axis such that PCP4 labeling often extended beyond the distal border of the DLZ into CA1. Finally, we found that, consistent with previous findings, mossy fibers innervate a subset of RGS14 positive neurons (~65%–70%) and that mossy fiber bouton number and relative size in CA2 are less than that of boutons in CA3. Unexpectedly, we did find evidence of some complex spines on apical dendrites in CA2, though much fewer in number than in CA3. Our results indicate that certain molecular markers may be better suited than others when defining the proximal and distal borders of area CA2 and that the presence or absence of complex spines alone may not be suitable as a distinguishing feature differentiating CA3 from CA2 neurons.Hippocampal area CA2 is a molecularly and functionally distinct region of the hippocampus that has classically been defined as the area with large pyramidal neurons lacking input from the dentate gyrus and the thorny excrescences (TEs) characteristic of CA3 neurons. A modern definition of CA2, however, makes use of the expression of several molecular markers that distinguish it from neighboring CA3 and CA1. Using immunohistochemistry, we sought to characterize the staining patterns of commonly used CA2 markers along the dorsal–ventral hippocampal axis and determine how these markers align along the proximodistal axis. We used a region of CA2 that stained for both Regulator of G‐protein Signaling 14 (RGS14) and Purkinje Cell Protein 4  as a reference. Here, we report that certain commonly used CA2 molecular markers may be better suited for drawing distinct boundaries between CA2/3 and CA2/1. For example, RGS14+ and STEP+ neurons showed minimal to no extension into area CA1 while areas stained with VGluT2 and  This finding was confirmed by Ishizuka et al., and others, with Lorente de Nó's nomenclature updated to include the absence or presence of thorny excrescences (TEs) characteristic postsynaptic structures associated with MF synapses  to label the specialized extracellular matrix called perineuronal nets , basic fibroblast growth factor (bFGF/FGF2), and alpha‐actinin 2 had been noted to be enriched in CA2  or effector proteins using Cre recombinase under the control of CA2‐specific promoters like Given the myriad of molecular tools available, we set out to further investigate the morphological features associated with the MF CA2 synapses and the distinct molecular characteristics of hippocampal area CA2, in relation to the neighboring CA subfields, to better understand both how best to define cellular heterogeneity in this region. In addition, we sought to gain a clearer understanding of the degree to which commonly used CA2 immunohistochemical markers colocalize in the proximal and distal borders of hippocampal area CA2.22.1All procedures were approved by the NIEHS Animal Care and Use Committee and were in accordance with the National Institutes of Health guidelines for care and use of animals. Wild‐type adult mice, either c57BL/6J (Jax 000664) or bred on a c57BL/6J background, were used for all experiments. Male and female adult mice (13–22 weeks old) were used for immunofluorescence experiments. Both male and female adult mice were used for viral injection experiments.2.2Adult mice were temporarily anesthetized using isoflurane (0.5% at 0.8 L/min) and then received and intraperitoneal injection of a ketamine/xylazine cocktail  at 0.1 cc/10 g body weight. Ophthalmic ointment was then applied to the eyes and the scalp was shaved and treated with two applications of betadine. Lidocaine (0.05 cc of 0.5% Marcaine) was injected subcutaneously. After being secured in the stereotaxic instrument, an incision was made along the midline exposing the skull surface and after removing the dura, pilot holes were unilaterally drilled above area CA2  and DG . Virus was infused into either CA2  or into the DG  using a Hamilton syringe position in a syringe pump (KD Scientific) and tube‐fed cannulas made in house from blunted 26G needles (BD). The cannula was left in position for a minimum of 8 min before a slow retraction. The scalp was then sutured, and Buprenorphine‐SR was administered subcutaneously  followed by the antisedant Atipamezole . Animals were allowed to recover on a warming pad until regaining consciousness and returning to the home cage. Animals remained in their home cages for a minimum of 2.5 weeks to allow for the proteins to express prior to tissue collection.2.3Mice were fully anesthetized with Fatal Plus  via intraperitoneal injection. Immediately after transcardial perfusion with 4% paraformaldehyde (pH 7.4) in phosphate buffered saline (PBS), brains were removed and post‐fixed in the same fixative overnight at 4°C. Brains were sectioned at 40 μm, at an angled horizontal plane .The following antibodies were used to label RGS14 and PCP4 in most experiments: mouse anti‐RGS14 , rabbit anti‐PCP4 , Alexa Fluor 568 secondary , and Alexa Fluor 488 secondary . Antibodies not mentioned here are summarized in Table 2.4t test assuming equal variance and a Gaussian distribution. If distributions proved to be unequal, a Welch's t test was calculated and reported as such in the text. Data are presented as means ± SEM.All images were acquired with an LSM 880 Zeiss confocal microscope. All quantifications were made on maximum intensity Z‐projected images with ImageJ  in CA1‐3. Dorsoventral depths corresponding approximately to the following figures from Paxinos and Franklin's mouse brain atlas  and other commonly used markers of CA2, 3D tile scans  were obtained at 0.35 μm (Z‐depth) increments to include all labeled RGS14 and PCP4 neurons of the stratum oriens (SO) border of distal CA1 is too diffuse to delineate objectively, the width of the SP was restricted throughout the more distal CA1 to match the width of the widest portion of the CA1 compact pyramidal cell layer.RGS14+/PCP4− and PCP4+/RGS14− cells were observed along the proximodistal hippocampal axis. Continuous immunohistochemical staining was defined as positively labeled cells being no more than 200 μm apart. Accordingly, imaging windows included at least 200‐μm beyond each border of continuous labeling. Measurements of each individual stain  were made using a magnification level of 150%, by an investigator blinded to just one stain. All counts for continuously labeled cell bodies were restricted to the SP and measured along the midline of SP in CA1‐3. We defined the CA1‐subiculum border as the distal‐most end of the compact pyramidal cell layer. Because the SP‐To determine the RGS14+/PCP4+ DLZ, the overlap of RGS14 and PCP4 was then visually inspected for co‐expression. Within the length of RGS14/PCP4 overlap, the borders of the DLZ were defined by the distal‐most and proximal‐most cells that show double‐labeling of RGS14 and PCP4.stratum lucidum. ImageJ was used to measure the area (μm2) of each hand‐drawn MFB. Large MFBs were characterized by a cross‐sectional area of at least 4 μm2, equivalent to the minimum volume (6 μm3) characteristic of giant MFBs  within the distal‐most 50 and 100 μm of the SL.For quantification of mossy fiber boutons (MFBs), 3D Z‐series stacks  were obtained at 0.64‐μm increments along the SL of the CA2‐CA3 axis. Two dorsal and two intermediate sections  each from all three animals were imaged 8.5‐μm thick maximum intensity projections centered on the SL of CA2 were generated and quantified at a magnification level of 75% in ImageJ. All mCherry‐positive MFBs within the SL were drawn manually, along the midline of the SL, as a function of distance from the distal end of 33.1Early investigations into hippocampal neuronal morphology by Lorente de Nó defined area CA2 as distinct region of Cornu Ammonis (CA) characterized by pyramidal neurons that lacked innervation from MF axons originating from granule cells of the DG, as well as an absence of complex spines, or thorns/TEs, seen on apical dendrites in CA3 neurons , towards CA1, and analyzed using ImageJ software , with a 166% reduction in boutons when comparing the CA3 region 500–600 μm from dSL, towards CA3, with the 100 μm adjoining the dSL . Last, identified boutons were divided into small (<2 μm2), medium (≥2 μm2 <4 μm2), and large (≥4 μm2) groupings. All three categories were equally represented in the distal‐most 100 μm of SL  with a large/small ratio of 1.2/1 , while a second injection containing (non‐Cre dependent) GCamp6f to label both CA2 and distal CA3 (CA3d) pyramidal neurons was targeted to rostrodorsal area CA2 Figure . MFBs weml-neuronbrowser.janelia.org). To further characterize the individual boutons of these distinct projections, we again labeled both presynaptic boutons (red) and postsynaptic neurons (green) and identified individual boutons in the horizontal plane  but then fell off dramatically in the more proximal regions of CA3 in the more ‘intermediate’ sections ventral to the viral injection site  in both dorsal sections  or mean bouton density . We note that no boutons were observed in CA3 at distances >400 μm from dSL in intermediate sections ventral to the viral injection site.Previous work has described an interesting phenomenon wherein MF axons originating from dorsal granule cells project in the horizontal plane to contact nearby CA3 and CA2 neurons but then continue ventrolaterally within CA2 and seem to target area CA2 exclusively . On average, the RGS14+ overlap with Znt3+ stratum lucidum measured 316.24 ± 7.58 μm in dorsal sections, which was significantly greater than in ventral hippocampus . Interestingly, the percent overlap of the two markers across the D‐V axis was similar at both levels implying that reduction in total RGS14 length is, on average, proportional to the reduction in RGS14+/Znt3+ overlap in dorsal and ventral sections . The remaining ~30% of RGS14+ neurons did not overlap with Znt3+, marking a subset of CA2 neurons that are unlikely to receive direct synaptic input from granule cells, and this fraction was also significantly greater than in ventral sections .In an effort to further characterize the relationship between MF axons and area CA2, we used immunohistochemistry (IHC) to label Zinc transporter 3 (Znt3) positive MFs and RGS14 positive neurons in both dorsal and ventral horizontal hippocampal sections Figure . When mestratum lucidum. Our interpretation of the imaged location was further aided by the RGS14+ measurements reported in Figure stratum lucidum towards CA3, though such excrescences uncommon  was that a single injection labeled both “off‐target” CA3 and targeted CA2 neurons, allowing us to visualize postsynaptic morphologies along the proximodistal CA3 → CA2 axis (towards CA1), including cells that both were and were not aligned with n Figure . Althoug3.2p = .006, dorsal vs. ventral; 526.04 ± 15.75 μm, p = .033, n = 59, 44, 42 sections from 17, 15, 13 animals, respectively). Conversely, PCP4+ labeling significantly increased along the same axis with a mean length of 648.98 ± 21.42 μm in dorsal hippocampus, 774.23 ± 38.44 μm in intermediate sections and 852.96 ± 35.46 μm in ventral sections . Interestingly, the region of overlap, or DLZ, between these two CA2 markers, was significantly greater in dorsal sections compared with intermediate and ventral sections, similar to RGS14+ measures reported in Figure n = 51, 44, 42 sections, from 13 animals). While quantifying this overlap, we observed a pattern wherein these markers labeled neurons beyond the double labeled region, and so we aimed to quantify the extension of these hallmark CA2 markers beyond the DLZ in the direction of neighboring CA1  the degree to which RGS14 labeling colocalizes with another hallmark label of CA2, PCP4, and (2) whether that colocalization is dependent on dorsal–ventral location. For these experiments, we performed IHC to label both RGS14 and PCP4 in dorsal, intermediate, and ventral horizontal sections  though, at both levels, we found a higher percentage of quantified sections exhibiting RGS14+ staining beyond the DLZ and into CA3 . In ventral hippocampus, RGS14+/PCP4− straggler cells were less common and observed in only 26% of analyzed sections versus in 36% of sections with PCP4+/RGS14− cells . Taken together, we find reliable and continuous colocalization of RGS14 and PCP4 labeling across the dorsal–ventral axis, consistent with previous work, but observe heterogeneity among individually labeled neurons that extend beyond this double labeled region. Although RGS14 aligns well with the DLZ at the putative CA2/1 border, PCP4+ neurons were found to extend well into CA1 at all three DV levels and most abundantly at more ventral depths. Conversely, although we found both RGS14+ and PCP4+ neurons extending beyond the CA2/3 end of the DLZ, RGS14 staining was more likely to extend at both dorsal and intermediate depths and PCP4 being more likely to extend into ventral CA3.Regarding neuronal labeling proximal to the DLZ, towards CA3, we observed instances in which both RGS14+/PCP4‐ and PCP4+/RGS14− “straggler cells” extended beyond the DLZ. We found no significant difference in the mean length of these extensions of either labeled populations at any of the dorsoventral depths measured , though the difference was observed in dorsal‐most sections as well . Furthermore, the mean length of WFA staining is significantly longer in dorsal sections versus intermediate and ventral . This pattern is not biased to either the proximal (CA3) or the distal (CA1) end of the DLZ (data not shown). Given the fact that WFA is also known to label interneurons in the region, cells that were WFA but RGS14−/PCP4− were not included in WFA+ length measurements. Similarly, the mean length of immunopositivity for the PNN component ACAN was also significantly shorter at dorsal depths when compared with RGS14+ . Interestingly, when CA2 neurons were labeled with the fluorescent reporter GCaMP6f in combination with IHC staining of WFA and Znt3 , a region that we showed earlier was RGS14+ . This finding is most pronounced in intermediate and ventral sections  and the Vesicular Glutamate Transporter 2 (VGluT2) contribute to the distinct molecular profile of the CA2 region and label CA2 soma and SuM presynaptic terminals, respectively , the mean length of Amigo2‐GFP labeling is significantly longer than that of the DLZ in ventral levels  but extends beyond the DLZ in more ventral sections . With respect to the CA2/3 border region, GFP positive neurons extending beyond the CA3 border of DLZ are observed at all levels and increase in length along the dorsal‐intermediate‐ventral axis . This was especially true with respect to PCP4 extension into CA1, which as we have shown, increases in likelihood in more intermediate and ventral hippocampal sections.The CA2‐specific enrichment of the cell‐adhesion protein Amphoterin‐induced gene and open reading frame 2 (AMIGO2) has been employed by many labs as a genetically encoded marker that can be used to not only distinguish CA2 from CA1 and CA3 but also to manipulate CA2 neuronal activity in vivo during specific behavioral tasks  and medium (≥2 μm2 < 4 μm2) boutons remains relatively consistent along the CA2 → CA3 disto‐proximal axis we observe large MFBs becoming the predominate fraction beginning in the bin 200–300 μm from dSL and continuing through CA3 proper in more dorsal hippocampal sections. We also report that RGS14‐positive neurons extend, on average, ~300 μm from the end of stratum lucidum towards area CA3. Therefore, it is possible that the large MFBs that we observe in the 200 300 μm bin form synapses onto CA2 neurons near the CA2/CA3 interface. This idea that MF synapses in CA2 can in fact be comprised of presynaptic and postsynaptic compartments with morphologies similar to that of the much‐studied CA3 synapses is further supported by high resolution, high magnification confocal images that clearly show large, complex postsynaptic spines in stratum lucidum at distances <200 μm from the distal most end of the MF pathway  or CA2 neurons (Amigo‐2) in concert with immunohistochemical CA2 markers to build upon our reported characterization of complex spines along the apical dendrites of hippocampal pyramidal neurons.Consistent with previous work . Future work will also continue the characterization of immunohistochemical markers like STEP and ACAN to better understand how these markers align with the DLZ in more intermediate and ventral hippocampal sections.Immunohistochemical markers are valuable tools to label area CA2 in ex vivo and in vitro preparations but how consistently do they label the same population of neurons and how do these patterns persist along the dorsal–ventral hippocampal axis? To answer these questions, we compared the proximodistal lengths of commonly used molecular markers of CA2 to the region of RGS14+ and PCP4+ colocalization (DLZ) along the dorsal–ventral axis to gain insight into potentially useful patterns of expression. Throughout our analysis, RGS14 and STEP reliably labeled the distal end of the DLZ with little to no evidence of extension into CA1. PCP4+ staining, on the other hand, consistently extended beyond the DLZ and into CA1 with this observed pattern increasing in scale along the DV axis. Conversely, WFA, ACAN, and VGluT2 positive staining fell significantly short of the distal DLZ at all hippocampal depths. Taken together, PCP4, WFA, ACAN, and VGluT2 exhibit legitimate caveats if being used to confirm the border between areas CA2 and CA1. On the more proximal end of the DLZ, our analysis shows that VGlut2+ positive staining is useful for distinguishing the CA2/CA3 border in dorsal and intermediate sections but is less reliable in more ventral sections. Conversely, staining for STEP extended beyond proximal DLZ and into CA3 with a mean extension length of 247.4 ± 72 μm, while WFA staining mean length fell short of the proximal DLZ border at all measured depths. RGS14 and PCP4 exhibit more variability at the CA2/3 border with respect to position along the DV axis. Though both markers showed similar mean lengths of extension beyond the proximal DLZ, though in separate cells, PCP4 extension was observed in a lower percentage of sections when compared with RGS14 in dorsal and intermediate levels, while the opposite pattern was observed in ventral levels where RGS14 was less likely to extend into CA3 relative to PCP4. We also report that when using an Am2‐GFP mouse line to label CA2 neurons, we observed GFP signal extending beyond the DLZ. Furthermore, these GFP+ neurons were most commonly PCP4+/RGS14− though, in a few instances, we observed GFP+ neurons that were negative for both RGS14 and PCP4. While this may be an interesting finding, we note that, more likely, antibody penetration into the tissue is the limiting factor when comparing IHC staining with the expression of a genetically expressed marker, which will be seen throughout the tissue thickness. Further work would be necessary to confirm any lack of IHC‐derived signal in GFP+ neurons as an indicator of AMIGO2  Lateral view of hippocampus indicating dorsal, intermediate, and ventral depths for analysis. (a1–3) DAPI‐stained horizontal sections showing dentate gyrus anatomies of serial sections corresponding to dorsal (1), intermediate (2), and ventral (3) depths in (a). Dorsoventral depths correspond approximately to interaural depths  of Paxinos and Franklin's horizontal mouse brain atlas (2012).Click here for additional data file.FIGURE S2: Mossy fiber bouton (MFB) increase in number but not mean density along the distoproximal CA2 → CA3 axis. (a1) Reference overview image  of a dorsal horizontal section showing GCaMP6f‐expressing CA2/CA3 pyramidal neurons and mCherry‐expressing mossy fibers (MFs) originating from dorsal dentate gyrus. (a2–3) Expanded views  of insets from  showing hand‐drawn boundaries of isolated, well‐defined boutons in the distal‐most 0–100 μm of SL  and 500–600 μm from dSL . Inset in  shows example of a typical MFB in CA3. Scale bar = 3.5 μm (b) Total number of MFBs increases along the CA2 → CA3 axis of dorsal hippocampus . (c) Total MFB density in the 0–100 μm bin (relative to the end of stratum lucidum in CA2) is statistically similar in CA2 and CA3 .Click here for additional data file.FIGURE S3: Some CA2 pyramidal neurons display thorny excrescences on apical dendrites in stratum lucidum.  Z‐projected confocal images  of horizontal sections from dorsal hippocampus showing GCaMP6f‐expressing CA2 pyramidal neurons and mCherry‐expressing mossy fiber boutons originating from dorsal dentate gyrus granule cells. Apical dendrites of select CA2 pyramidal neurons display thorny excrescences (white arrows) located within 160 μm (a) and 90 μm (b) from dSL, respectively. Yellow asterisks indicate apical dendrites that lack thorny excrescences. Inset: Enlarged view showing thorny excrescences of a MF‐CA2 synapse.Click here for additional data file.FIGURE S4: Purkinje Cell Protein 4 (PCP4+)/Regulator of G‐protein Signaling 14 (RGS14−) extension beyond CA1 border of double‐labeled zone (DLZ) is most common in ventral levels.  Z‐projected confocal images showing PCP4 neuronal labeling in CA1 of horizontal sections from dorsal (a) and ventral (b) hippocampus. (c) Percentage of sections exhibiting PCP4+/RGS14− labeling beyond DLZ and into CA1 is largest in ventral hippocampus . (d) In the dorsal and intermediate hippocampus, the percentage of sections that exhibit RGS14+/PCP4− labeling beyond the DLZ, into CA3, is larger than the percentage of sections that exhibit PCP4+/RGS14− labeling in CA3. RGS14+/PCP4− straggler cells in CA3 are rare in the ventral hippocampus .Click here for additional data file.FIGURE S5: Dorsoventral differences in the labeling pattern of VGluT2. (a) VGluT2 labeling in the stratum oriens extends diffusely into proximal CA3 in ventral (right), but not dorsal (left) depths. (b) The mean fluorescence intensity of VGluT2 labeling in stratum oriens is greater in ventral levels . Scale bars = 100 μm.Click here for additional data file.