Although AMR strains arise naturally due to genetic changes in replicating microorganisms, their emergence is accelerated by inappropriate use of antimicrobial agents in humans, animals, and the environment.12 In 2016, African countries committed to address AMR as part of the United Nations General Assembly special meeting and, in June 2017, committed to accelerate implementation of the International Health Regulations and promote Africa’s health security, of which AMR surveillance and control is a core component.14 To implement these commitments, the Africa Centres for Disease Control and Prevention (Africa CDC) officially launched its ‘Framework for Antimicrobial Resistance Control, 2018–2023’ in October 2017.15 Established in January 2017, Africa CDC is a ‘specialised technical institution’ of the African Union (AU) responsible for strengthening capacity for surveillance and disease intelligence, emergency preparedness and response, laboratory systems, information systems, and public health research and workforce development in Africa.16Recognising the urgent need for action, the World Health Assembly adopted the Global Action Plan on Antimicrobial Resistance in May 2015, and the World Health Organization established the Global Antimicrobial Resistance Surveillance System.Africa CDC developed its framework with four principles in mind. Firstly, the strategy should help advance the Global Action Plan. Secondly, while more information is needed about incidence, prevalence, and interventions, Africa CDC should follow the ‘precautionary principle’ and take action against resistance now. Thirdly, policy and practice are as important as diagnostic or treatment technologies in mitigating AMR. Finally, Africa CDC has an opportunity to leverage its unique position in the AU to raise awareness, secure commitments, and influence policy at the head of state level. After consultation with subject matter experts in Africa and globally, Africa CDC developed a framework that has four objectives: (1) improve surveillance of AMR organisms among humans and animals, (2) delay emergence of AMR, (3) limit transmission of AMR and (4) mitigate harm among patients infected with AMR organisms. 3 Although gains have been made in collecting data on resistance to some pathogens, such as HIV, Mycobacterium tuberculosis, and Plasmodium spp., several challenges remain, including inadequate demand by clinicians for diagnostic testing, laboratory infrastructure, resources to continuously collect, transport, and test specimens for AMR surveillance, use of standardised protocols, quality assurance, systematic surveillance of AMR in animals and their products in Africa, and collaboration between the human and animal health sectors.18 Africa CDC’s framework identified several areas in which it can strengthen laboratory capacity for surveillance in humans and animals.Understanding the full extent of AMR and its impact in Africa is challenged by a lack of continent-wide AMR surveillance data, especially for pathogens that require complex testing methods.19 Success has been achieved when stewardship programmes include structural changes and behavioral modification techniques.22 Economic incentives for prescribing vary by country but can be particularly problematic when physicians or health care facilities rely on antimicrobial sales to fund operations. In many settings, persons obtain antimicrobials directly from pharmacies with no evaluation by or prescription from a physician.23 Additionally, substandard and counterfeit antimicrobials are widely available in Africa; substandard antimicrobials can promote AMR by containing levels of an agent sufficient to exert selective pressure for resistant organisms but insufficient levels to kill those organisms.9 There are limited studies about the effectiveness or essential components of antimicrobial stewardship programmes in Africa and other resource-limited settings, particularly involving the engagement of non-physician prescribers.24Reducing the need for and inappropriate use of antimicrobials can help delay emergence of AMR. In human health, extensive studies have been conducted in the United States, Europe, and other high-income settings on approaches to promote prudent antimicrobial prescribing – an approach known as antimicrobial stewardship.26 The AU has made a high-level commitment to food safety, engaging partners from multiple sectors, including the industry. Africa CDC can leverage this commitment by making policymakers more aware of the link between food production, AMR emergence, and food-borne illness and by promoting prudent antimicrobial use in collaboration with animal health, agriculture, and other related authorities.In animals, antimicrobials are used for prophylaxis, treatment, and growth promotion, and it is reasonably assumed that most antimicrobial consumption occurs during food production, not through prescriptions to treat illness. Prudent use in these settings is a critical risk management approach to delaying emergence, but is challenging, because food producers have a strong economic incentive to use antimicrobials and weak incentive to use them prudently.27 Such transmission can have severe consequences, because pathogens in health care facilities are more likely to be multi-drug resistant. Hospitalised people are more susceptible to these severe illness, and these pathogens can also be spread outside of the hospital. The basic components of all programmes include: strong political commitment and dedicated resources for infection control, strict adherence to protocols for hand hygiene and for identification, isolation, and management of potentially infectious patients, adequate supplies and equipment for patient care, systems for infectious waste management, building design, and maintenance to reduce transmission, and continuous monitoring of process, outcome, and impact indicators.28 The West Africa Ebola response demonstrated that effective hospital-based infection prevention and control programmes require intensive support to initiate and sustain. Africa CDC’s framework, therefore focuses primarily on advocacy, policy, and guidance to mobilise sufficient resources for infection prevention and control.Transmission of AMR occurs frequently in health care facilities.29In animal health, implementation of risk-based food safety systems – such as hazard analysis of critical control points, good handling practices, and good agricultural practices – can reduce the flow of AMR pathogens in food to humans, especially when combined with proper nutrition to reduce the need for antimicrobials.30 Substandard diagnostic tests and laboratory supplies remain common problems across Africa and can lead to AMR when tests lead to unnecessary antibiotic treatment. Limiting their availability will be challenging, given the large number of health care delivery sites that may be using such products and the insufficient supply of quality diagnostic tests. A major ethical dilemma for Africa CDC’s framework will be to balance antibiotic access versus excess.31 Although antibiotic usage is excessive at a population level, many vulnerable groups lack access to effective antimicrobial treatment. Africa CDC’s framework focuses on promoting antimicrobial and diagnostic stewardship, while seeking ways to maintain access to life-saving diagnostics and treatments.Strong antimicrobial stewardship programmes promote adherence to clinical treatment guidelines, helping delay emergence of AMR and improve outcomes among patients already infected with AMR organisms. Diagnostic stewardship will also be essential to guide such treatment.Laws and policies play a critical role in framing, enabling, and protecting public health. Africa CDC will leverage its stature and authority as a specialised agency of the AU to advocate for laws and policies to monitor, prevent, and mitigate AMR. Such policy initiatives, however, can only succeed with robust involvement of civil society. To date, engagement of civil society for AMR has been challenging, because the science can be complex to explain, the threat often characterised as distant, patients’ stories of illness and death often not told because of under-diagnosis and because they occur in marginalised groups, interventions not readily distilled into high-impact slogans, and public health agencies investing too little in civil society engagement.To effectively control AMR, each country will need leadership and staff that have the education and skills to implement AMR control programmes in both human and animal health sectors. The most pronounced human resources gaps are likely in laboratory services – from bench microbiologists to managers – and in health care infection prevention and control. Africa CDC and partners are working together on a public health workforce strategy for the continent and mobilising resources to support hiring, training, and retaining of staff. By highlighting the urgency and severity of the AMR threat, Africa CDC will advocate for both donors and government ministries to allocate more funding to laboratory services and infection prevention and control personnel.From 4–5 April 2018, Africa CDC convened AMR focal points from African countries and partners to review the Africa CDC AMR framework and prioritise activities for the next year. One major conclusion of the meeting was that Africa CDC should establish a formal structure to coordinate AMR activities within the AU, particularly with the AU’s animal health agency , across member states, and with multilateral and non-governmental partners in Africa. Participants proposed that this entity’s first task should be to transform the Africa CDC AMR framework into an AU-wide framework and ensure that it more fully addresses AMR issues in animal and environmental health. In addition to governance and coordination, participants proposed that Africa CDC focus activities in the next year on developing and harmonising tools to map and assess human and animal diagnostic laboratory capacity for AMR, developing guidelines for prudent antimicrobial use in humans and in animals that reflect what is currently known about AMR in Africa, and developing minimum standards for infection prevention and control in health care facilities to limit AMR transmission.Africa CDC’s framework for AMR is broad and aspirational, reflecting the urgency and complexity of this public health problem and the potential for Africa CDC as an institution to help galvanise action and provide guidance to member states. Progress in implementing this framework will require close coordination across and high-level commitment from member states and partners in Africa.


A receptor within the ventral hippocampus (vHipp). Indeed, this is sufficient to normalize vHipp activity and downstream alterations in dopamine neuron function in the MAM rodent model. This approach also attenuated behavioral deficits in cognitive flexibility. To understand the specific pathways that mediate these effects, we used chemogenetics to manipulate discrete projections from the vHipp to the nucleus accumbens (NAc) or prefrontal cortex (mPFC). We found that inhibition of the vHipp-NAc, but not the vHipp-mPFC pathway, normalized aberrant dopamine neuron activity. Conversely, inhibition of the vHipp-mPFC improved cognitive function. Taken together, these results demonstrate that restoring GABAergic signaling in the vHipp improves schizophrenia-like deficits and that distinct behavioral alterations are mediated by discrete projections from the vHipp to the NAc and mPFC.Hippocampal hyperactivity is correlated with psychosis in schizophrenia patients and likely attributable to deficits in GABAergic signaling. Here we attempt to reverse this deficit by overexpression of the α5-GABA Region-specific increases in hippocampal activity have been reported in schizophrenia patients, yet there are little known about how these circuit levels changes modulate behavioral deficits. Here authors found that that over-expression of the a5 subunit of the GABAA receptor increased tonic GABA currents and normalized aberrant pyramidal cell activity in the ventral hippocampus which had distinct disease symptom outcomes depending on the pathway targeted Although the long-standing dopamine hypothesis of schizophrenia suggests that hyperactivity in the mesolimbic dopamine system contributes to disease symptoms4, antipsychotic medications, which act as antagonists at the dopamine D2 receptor, are only somewhat effective in treating positive symptoms and have little to no impact on cognitive symptoms of schizophrenia5. Further, schizophrenia patients do not display overt pathology in the mesolimbic dopamine system, leading some to suggest that the pathology of schizophrenia lies in upstream brain regions that regulate dopamine signaling6.Positive symptoms, such as delusions and hallucinations, are often the most striking features of schizophrenia; however, patients also display characteristic cognitive symptoms, such as working memory deficits and cognitive inflexibility, which can negatively influence social and occupational functioning and diminish quality of life7. This increase is correlated with positive symptom severity8 as well as cognitive dysfunction9, suggesting that exaggerated hippocampal activity may be a key pathogenic factor in schizophrenia. The vHipp hyperactivity observed in schizophrenia patients is thought to result from a deficit in GABAergic inhibition9. For example, schizophrenia patients show cell loss restricted to specific GABAergic interneuron subtypes  in the hippocampus11. Previously, we demonstrated that gestational exposure to the mitotoxin methylazoxymethanol (MAM) produces anatomical, physiological, and behavioral deficits that model schizophrenia [for review see ref. 12], including a loss of hippocampal interneurons13 and corresponding hippocampal hyperactivity14, and behavioral correlates of positive14, negative15, and cognitive symptoms15. We further demonstrated that transplanting interneurons derived from embryonic stem cells, normalizes hippocampal activity and attenuates behavioral correlates of positive and cognitive symptoms in the MAM model15. One way in which GABAergic interneurons regulate the function of pyramidal cells in the hippocampus is via the ionotropic GABAA receptor, a heteropentameric chloride ion channel. The α5 subunit of the GABAA receptor is unique in its relatively limited distribution within the hippocampus16. This subunit has been shown to regulate the timing of pyramidal cell firing, action potential thresholds, and coordinated oscillatory activity17. Interestingly, hippocampal specific knock-down of the α5 receptor subunit produces impairments that recapitulate positive symptoms of schizophrenia, including deficits in latent inhibition and pre-pulse inhibition19. Conversely, systemic α5 agonists normalize dopamine signaling and improve behavioral correlates of positive symptoms in rodent models of schizophrenia20, suggesting that this subunit of the GABAA receptor may be a viable therapeutic target. However, it is unclear whether enhancing signaling at the α5 GABAA subunit would also improve cognitive symptoms, which are poorly treated by currently prescribed antidepressants.The hippocampus is one region where functional and anatomical changes have been consistently observed in schizophrenia patients. One of the more reliable observations in schizophrenia patients is increased hippocampal activity at rest21. In the current experiments, we use viral-mediated gene transfer to restore inhibitory signaling in the vHipp by over-expressing the α5 subunit of the GABAA receptor in pyramidal cells. We found that α5 overexpression increased tonic GABA currents and normalized aberrant pyramidal cell activity in the vHipp. This approach also normalized aberrant dopamine signaling and cognitive function in a rodent model of schizophrenia, suggesting this may be a promising novel treatment strategy for schizophrenia. Next, we identified the neural circuit mechanisms underlying these effects. Using chemogenetics, we identified the discrete pathways from vHipp that mediate behavioral and physiological deficits that mirror positive and cognitive symptoms of the disorder. Together, these experiments identify a novel approach for treating schizophrenia and provide insight into the anatomical and neurochemical pathways associated with discrete dimensions of schizophrenia, so that therapeutics can be developed with improved efficacy for treating multiple symptom domains.Gene therapy holds the promise of treating diseases by replacing defective genes and has been used to target GABAergic signaling in neurological disorders22. Therefore, we used whole-cell patch-clamp recordings to determine if α1 or α5 overexpression was sufficient to augment tonic or phasic activity. We found that over-expressing the α5 subunit in pyramidal cells of the vHipp increased tonic GABA currents without affecting phasic activity. Specifically, we found that α5 overexpression increased the amplitude of tonic GABA currents  = 6.84, p < 0.05; Holm–Sidak GFP vs α5 t = 2.51, p < 0.05; GFP = −19.47 ± 4.29 pA, α5 = −44.16 ± 10.84pA; n = 6–9 cells per group) while overexpression of the α1 subunit had no effect on tonic current amplitude . Neither IPSC amplitude nor frequency were affected by either α1 or α5 overexpression in hippocampal slice preparations  = 0.30, p > 0.05; Holm–Sidak GFP vs α1 t = 0.07, p > 0.05; Holm–Sidak GFP vs α5 t = 0.67, p > 0.05; GFP = −54.07 ± 4.80 pA, α1 = −53.40 ± 4.78 pA, α5 = −60.31 ± 10.45 pA; IPSC frequency: one-way ANOVA F = 0.17, p > 0.05; Holm–Sidak GFP vs α1 t = 0.58, p > 0.05; Holm–Sidak GFP vs α5 t = 0.16, p > 0.05; GFP = 1.05 ± 0.28 Hz, α1 = 0.83 ± 0.30 Hz, α5 = 0.98 ± 0.21 Hz). Representative traces are shown in Fig. All data are presented as mean ± SEM. First, to confirm stable transgene expression in the vHipp, we use immunohistochemistry for GFP. As shown in Figure A receptor produces functional changes in in vivo vHipp pyramidal cell activity using extracellular electrophysiology. Consistent with the in vitro data, we demonstrate that α5, but not α1, overexpression normalizes the aberrant vHipp activity observed in the MAM model  = 4.73, p < 0.05; Prenatal Treatment F = 0.10, p > 0.05; Gene Therapy F = 2.40, p > 0.05; n = 43–62 cells per group). Specifically, as we have seen previously15, MAM-treated rats have an increase in pyramidal cell firing rate in the vHipp compared to saline controls . In MAM-treated rats that received the virus to over-express the α5 subunit of the GABAA receptor, this effect was completely abolished . In saline-treated rats, α5 overexpression had no effect on pyramidal cell firing rate in the vHipp . Overexpression of the α1 subunit had no effect in either the saline- or the MAM-treated animals . Representative electrophysiology traces are depicted in Fig. Next, we determined if overexpression of the α1 or α5 subunit of the GABA6. Therefore, we used in vivo extracellular electrophysiology to measure dopamine cell activity as a proxy for positive symptoms and found that overexpression of the α5 subunit of the GABAA receptor in the vHipp can normalize aberrant dopamine population activity in the VTA  = 12.43, p < 0.05; Prenatal Treatment F = 19.18, p < 0.05; Gene Therapy F = 24.25, p < 0.05; n = 6–7 rats per group). As we have shown previously15, MAM-treated animals have an increase in the number of spontaneously active dopamine cells per track compared to saline-treated control animals . This effect is attenuated in MAM-treated animals when the α5 subunit of the GABAA receptor is over-expressed in pyramidal cells of the vHipp . Dopamine cell population activity was not affected by α5 overexpression in saline-treated controls . We also analyzed two additional parameters of dopamine cell activity: firing rate and the percentage of action potentials fired in bursts. We found that overexpression of the α5 subunit in the vHipp affected firing rate  = 2.522, p > 0.05; Prenatal Treatment F = 0.05, p > 0.05; Gene Therapy F = 4.52, p < 0.05). In the saline-treated animals, the α5 overexpression produced a significant decrease in dopamine cell firing rate compared to animals that received the control virus . In the MAM-treated animals, gene therapy had no effect on the firing rate of dopamine neurons . Overexpression of the α5 subunit of the GABAA receptor in the vHipp had no effect on the bursting activity of dopamine neurons in the VTA  = 0.52, p > 0.05; Prenatal Treatment F = 0.06, p > 0.05; Gene Therapy F = 2.35, p > 0.05; saline/GFP = 36.97 ± 5.45% bursting; saline/α5 = 32.89 ± 4.61% bursting; MAM/GFP = 39.33 ± 4.98% bursting; MAM/α5 = 28.05 ± 4.98% bursting). Representative traces are shown in Fig. A receptor in pyramidal cells of the vHipp normalizes activity in the dopamine system and may attenuate the dopamine-related positive symptoms of schizophrenia.Although it is difficult to model delusions and hallucinations in a rodent, these positive symptoms have been attributed to increases in dopamine neurotransmission23. Using the attentional set-shifting test, we demonstrated that MAM-treated animals also show a deficit in reversal learning, which was not affected by gene therapy  = 0.04, p > 0.05; Prenatal Treatment F = 26.28, p < 0.05; Gene Therapy F = 0.62, p > 0.05; n = 5–7 rats per group). Specifically, we found that MAM-treated animals show an increase in trials to meet criterion compared to saline-treated controls . The MAM-induced deficit on reversal learning was not attenuated by overexpression of the α5 subunit of the GABAA receptor . Further, α5 overexpression had no effect on reversal learning in the saline-treated group .Reversal learning is one form of cognitive flexibility that is disrupted in schizophrenia23, a higher order form of cognitive flexibility that is critically dependent on the medial prefrontal cortex (mPFC)24. Unlike reversal learning, we found that gene therapy to over-express the α5 subunit of the GABAA receptor improves schizophrenia-like deficits in extradimensional set-shifting as measured by the attentional set-shifting test  = 6.02, p < 0.05; Prenatal Treatment F = 2.92, p > 0.05; Gene Therapy F = 2.97, p > 0.05; n = 5–7 rats per group). As we have shown previously15, MAM-treated animals have a deficit in extradimensional set-shifting as evidenced by an increase in trials to meet criterion compared to controls . This deficit was completely abolished in the animals that received gene therapy to over-express the α5 subunit in pyramidal cells of the vHipp . The α5 overexpression had no effect in the saline-treated controls . No other stages of the AST were affected by MAM treatment or α5 overexpression . In line with our previous findings, MAM-treated animals show a significant increase in the number of spontaneously active dopamine cells per track . Chemogenetic inhibition of the vHipp-NAc pathway attenuated the increase in dopamine population activity in MAM-treated animals but had no effect in controls . The firing rate  = 1.161, p > 0.05; Prenatal treatment F = 0.52, p > 0.05; DREADD condition F = 0.59, p > 0.05; saline/GFP = 2.6 ± 0.61 Hz, saline/Gi = 3.73 ± 0.61 Hz; MAM/GFP = 3.70 ± 0.61 Hz, MAM/Gi = 3.51 ± 0.61 Hz) and burst pattern  = 1.74, p > 0.05; Prenatal treatment F = 0.10, p > 0.05; DREADD condition F = 0.65, p > 0.05; saline/GFP = 65.83 ± 11.12%, saline/Gi = 42.22 ± 11.12%; MAM/GFP = 47.58 ± 11.12%, MAM/Gi = 53.29 ± 11.12%) of VTA dopamine cells were not affected by either MAM treatment or pathway inhibition.We have previously demonstrated that the increase in dopamine population activity is directly attributable to a pathologically enhanced drive from the vHipp1,16 = 2.17, p > 0.05; Prenatal treatment F1,16 = 32.76, p < 0.05; DREADD condition F1,16 = 0.29, p > 0.05; n = 4–5 per group). Although MAM-treated animals showed a significant increase in the number of spontaneously active dopamine cells per track , inhibition of the vHipp-mPFC pathway did not normalize dopamine population activity . Firing rate was not affected by either prenatal treatment or pathway inhibition  = 1.508, p > 0.05; Prenatal treatment F = 1.62, p > 0.05; DREADD condition F = 0.03, p > 0.05; saline/GFP = 3.93 ± 0.51 Hz, MAM/GFP = 3.91 ± 0.51 Hz, Saline/Gi = 4.66 ± 0.51 Hz, MAM/Gi = 3.37 ± 0.51 Hz). There was a main effect of the Gi DREADD on the percentage of action potentials fired in bursts  = 0.55, p > 0.05; Prenatal treatment F = 0.87, p > 0.05; DREADD condition F = 4.79, p < 0.05; saline/GFP = 51.01 ± 7.38%, MAM/GFP = 52.41 ± 7.38%, Saline/Gi = 29.37 ± 7.38%, MAM/Gi = 41.74 ± 7.38%). Cannula placements are shown in Fig. Conversely, inhibition of the vHipp-mPFC pathway has no effect on dopamine cell firing in the VTA  = 1.68, p > 0.05; Prenatal treatment F = 10.11, p < 0.05; DREADD Condition F = 8.38, p < 0.05; n = 6–7 per group). Specifically, we found that MAM treatment produced a deficit in reversal learning, as evidenced by an increase in trials to meet criterion . The reversal learning deficit was attenuated by inhibition of the vHipp-NAc pathway . Inhibition of the vHipp-NAc pathway had no effect on reversal learning performance in the saline-treated rats .To identify the neural pathways involved in unique forms of cognitive flexibility, we used the attentional set-shifting test. We found that hyperactivity in the vHipp-NAc pathway is responsible for reversal learning deficits in the MAM model of schizophrenia  = 0.71, p > 0.05; Prenatal treatment F = 4.46, p < 0.05; DREADD condition F = 0.0806, p > 0.05; n = 6–7 per group). MAM-treated rats required significantly higher trials to reach criterion than saline-treated controls . The MAM-induced deficit was not altered by inhibition of the vHipp-mPFC pathway . Together, these results suggest that hyperactivity in the vHipp-NAc pathway, but not the vHipp-mPFC pathway contributes to the reversal learning deficits observed in the MAM model of schizophrenia.Conversely, we found that reversal learning was not affected by inhibition of the vHipp-mPFC pathway  = 0.02, p > 0.05; Prenatal treatment F = 10.49, p < 0.05; DREADD Condition F = 0.01, p > 0.05; n = 6–7 per group). As seen previously, we found that MAM-treated animals have a deficit in extradimensional set-shifting as evidenced by an increase in trials to meet criterion . Inactivation of the vHipp-NAc pathway had no effect on extradimensional set-shifting in either the saline- or MAM-treated groups .Unlike reversal learning, we found that extradimensional set-shifting deficits are not affected by inhibition of the vHipp-NAc pathway  = 5.99, p <  0.05; Prenatal treatment F = 5.54, p < 0.05; DREADD condition F = 3.45, p > 0.05; n = 6–7 per group). As expected, MAM-treated animals showed a deficit in extradimensional set-shifting . Although inhibition of the vHipp-mPFC pathway had no effect in saline-treated animals, it completely abolished the MAM-induced deficit in extradimensional set-shifting . Performance on all stages of the AST are shown in Fig. Conversely, hyperactivity in the vHipp-mPFC pathway is responsible for MAM-induced deficits in extradimensional set-shifting  is responsible for schizophrenia-like deficits in a variety of rodent models26. In the current experiments, we used a lentiviral vector to over-express the α5 subunit of the GABAA receptor. Lentivirus has also been used in PD patients to express tyrosine hydroxylase, GTP cyclohydrolase, and aromatic acid decarboxylase, three enzymes needed for dopamine synthesis, in striatal neurons. This treatment resulted in an improved motor score for up to 1 year27. Further, in the current experiments, we demonstrate that we can improve multiple symptom domains by targeting a single brain region, the vHipp, reducing the possibility of negative side effects. However, further investigation of the long-term safety will be required before this treatment strategy can be moved to the clinic.Gene therapy, the method of using a viral vector to insert a gene directly into a cell, has shown great promise in recent years to not just treat symptoms but to actually cure certain diseases. For example, in Parkinson’s Disease (PD), adeno-associated virus was used to over-express the GABA synthesizing enzyme, glutamic acid decarboxylase, in the subthalamic nucleus. The unilateral infusion reduced metabolism in the thalamus and ipsilateral motor and premotor cortices, which was correlated with improved clinical disability ratings. These effects were still apparent at 1 year and were not associated with severe side effectsA receptor. GABAA receptors are heteropentameric chloride ion channels with 19 known subunits . The α5 subunit is unique as its expression is primarily limited to the hippocampus16, making it an ideal target for specifically reducing the hippocampal hyperactivity associated with schizophrenia. Further, the α5 subunit has been identified primarily extrasynaptically in dendritic fields16, where it is thought to mediate tonic inhibitory currents28. Indeed, in the current experiments, we demonstrate that over-expressing the α5 subunit increases tonic inhibitory currents in pyramidal cells of the vHipp. Further, we show that α5 overexpression also decreases the firing rate of hippocampal pyramidal cells, which is not surprising as tonic inhibition is thought to coordinate spike timing of pyramidal neurons and balance excitation17. By over-expressing the α5 subunit of the GABAA receptor, we normalized pyramidal cell activity in the vHipp.In the current experiments, we used viral-mediated gene transfer to increase expression of the α5 subunit of the GABA29. Further, elevated dopamine levels have been observed in the striatum of schizophrenia patients, and these levels have been correlated to the severity of positive symptoms30. Drugs that increase dopamine signaling can induce psychotic episodes in schizophrenia patients31. The current results are in line with previous work demonstrating that administration of the α5 partial agonist, SH-053–2’F-R-CH3, normalizes the number of spontaneously active dopamine neurons and amphetamine-induced locomotor activity in MAM rats20. Further, others have shown that α5 knock-out mice exhibit deficits in sensorimotor gating and latent inhibition19. Together, this data suggest that increasing α5 expression in the vHipp may act through the dopamine system to alleviate positive symptoms of schizophrenia. It is important to note that in animal models of schizophrenia, positive symptoms seem to result from increases in dopamine signaling specifically in ventral regions of the striatum, including the NAc32. However, in human studies, imaging techniques, such as positron emission tomography, have demonstrated that the largest changes in dopamine signaling are actually observed in associative regions of the striatum33. The reason for this discrepancy is a current area of research that remains to be elucidated.In addition, we demonstrated that α5 overexpression normalizes VTA dopamine cell population activity in the MAM model. Hyperactivity in the dopamine system is thought to underlie the positive symptoms of schizophrenia. For example, antipsychotic medications that block dopamine D2 receptors effectively reduce positive symptoms of the disorder34 and striatal D2 receptor availability35. Dopamine depletion in the caudate nucleus impairs reversal learning36. Further, both pharmacological agonism37 and antagonism38 of D2/D3 receptors can disrupt reversal learning, suggesting that this type of cognitive flexibility requires tightly controlled dopamine signaling. Because gene therapy normalized dopamine cell activity, we expected to see an improvement in reversal learning performance. Conversely, we did find that our gene therapy strategy attenuated the schizophrenia-like deficit in attentional set-shifting, a higher order form of cognitive flexibility mediated primarily by the medial prefrontal cortex (mPFC)24. These results suggest that gene therapy to over-express the α5 subunit of the GABAA receptor in pyramidal cells of the vHipp may be a viable treatment strategy to target some cognitive symptoms of schizophrenia, which have a profound impact on daily function3 but are poorly treated by currently available antipsychotic medications5.In addition to dopamine cell activity, we also tested the ability of α5 subunit overexpression to improve schizophrenia-like deficits in cognitive function. We were surprised to find that α5 overexpression had no effect on reversal learning, one form of cognitive flexibility that is disrupted in schizophrenia. Reversal learning has been associated with dopamine signaling. For example, reversal learning performance is correlated with striatal dopamine synthesis capacity39. These decreases in volume are primarily limited to the anterior hippocampus40, the sub-region of the hippocampus that corresponds to the ventral hippocampus of rodents42. Further, unmedicated schizophrenia patients show increased hippocampal activity at rest7, an effect that is attenuated by antipsychotic treatment43. Work in multiple animal models of schizophrenia has also demonstrated hyperactivity in the ventral hippocampus. For example, vHipp hyperactivity has been observed in developmental14, genetic44, and pharmacological models 25of schizophrenia. Further, we and others have shown that reducing hippocampal activity by transplanting inhibitory interneurons can normalize positive and cognitive symptoms of the disorder44. In the current studies, we expanded on these findings to identify the neural circuits from the vHipp that influence specific aspects of schizophrenia-like behavior. The concept that unique symptom clusters can arise from disruptions in subcortical structures, which then lead to abnormal regulation of cortical activity and dopamine system function, has been previously proposed by O’Donnell and Grace45. In order to test this hypothesis by manipulating discrete pathways from the vHipp we used chemogenetics, a method in which small molecule chemical actuators specifically interact with engineered proteins to affect the activity of a cell, such as G-protein-coupled receptors (GPCRs)46. Specifically, we used the Designer Receptor Exclusively Activated by Designer Drugs (DREADD), hM4Di, a modified M4 muscarinic acetylcholine receptor that couples to the inhibitory Gi pathway when activated by clozapine-N-oxide, a highly selective, but pharmacologically inert, exogenous ligand47. The hM4Di DREADD has been shown to silence neurons by hyperpolarizing the cell via activation of G-protein inwardly rectifying potassium channels (GIRKs)48 and inhibiting presynaptic neurotransmitter release49. In the current experiments, we used an adeno-associated viral vector to over-express the inhibitory DREADD in cells of the vHipp, then injected CNO directly into the NAc or mPFC to specifically inhibit vHipp neurons that project to these regions. Although CNO is often administered systemically to inhibit activity in discrete brain regions, this pathway-targeting strategy has been used successfully by others50.In the current experiments, we chose to focus on the hippocampus as both in schizophrenia patients and in animal models of schizophrenia, the hippocampus has been shown to be a key site of pathology. Decreases in hippocampal volume have been observed in schizophrenia patients at the time of first episode and seem to progress throughout the course of the illness45. In rodents, the vHipp can regulate dopamine signaling via a polysynaptic pathway from the NAc to the ventral pallidum (VP), and ultimately to dopamine cells in the ventral tegmental area (VTA)51. Specifically, the vHipp regulates the dopamine neuron population activity, or the number of spontaneously active dopamine neurons, which has been correlated with dopamine efflux in the NAc32. Importantly, it has been shown that only spontaneously active dopamine neurons can respond to stimuli by firing in a bursting pattern52. In this way, the vHipp is thought to regulate the ‘gain’ of the dopamine system6. In the current studies, we demonstrate that hyperactivity in the vHipp-NAc pathway of MAM animals increases dopamine population activity in the VTA by showing a reduction in the number of spontaneously active dopamine cells per track after inhibition of the vHipp-NAc pathway.One way in which the vHipp may influence schizophrenia-like behavior is through its control of the dopamine system23. Furthermore, impacting the OFC-NAc pathway could occur either by normalization of vHipp-NAc activity or normalization of VTA-NAc activity. Further experiments are required to conclusively determine which specific pathways and neurotransmitters are associated with the effect of vHipp-NAc on reversal learning.Interestingly, we also found that inhibition of the vHipp-NAc pathway attenuates MAM-induced deficits in reversal learning. This is in contrast to the alpha 5 overexpression experiment, which also normalized dopamine cell activity in the VTA but did not improve reversal learning in MAM-treated rats. Therefore, it is unlikely that inhibition of the vHipp-NAc pathway in MAM-treated animals improved reversal learning by normalizing dopamine activity in the striatum. However, other neurotransmitter systems and brain circuits have been implicated in reversal learning, including serotonin signaling in the orbital frontal cortex (OFC)53, a brain region that has been implicated in the cognitive symptoms of schizophrenia54. Schizophrenia patients show reduced prefrontal cortex activation during working memory tasks, with activation levels correlated to performance55. Even schizophrenia patients that perform normally on working memory tasks show higher levels of PFC activation compared to controls, suggesting a deficit in efficiency56. Further, anatomical changes, such as reduced dendritic spine density have been observed the PFC of schizophrenia patients57. In line with these findings, we demonstrated that reducing hyperactivity in the vHipp-mPFC pathway improved attentional set-shifting performance in MAM-treated rats. This is not surprising as the mPFC has been implicated in this form of cognitive flexibility24.In addition to its projection to the NAc, the vHipp also projects directly to the prefrontal cortex58. These studies also demonstrated that tonic activation of the mPFC produced subtle changes in VTA dopamine neurons suggesting that the effects of mPFC inhibition on VTA dopamine neuron activity would only be observed during periods of cortical burst firing.In the current experiments, we did not observe an effect of vHipp-mPFC pathway inhibition on dopamine population activity. However, it should be noted that we have previously demonstrated that synchronous cortical burst firing of the mPFC can increase dopamine population activity in the VTA59 and we have recently demonstrated that the paraventricular nucleus of the thalamus can also regulate dopamine signaling via the NAc60. Further, the vHipp sends and receives many projections to and from other brain regions beyond the NAc and mPFC. For example, reciprocal connections exist between the basolateral amygdala (BLA) and vHipp61 and optogenetic inhibition of this BLA-vHipp pathway has been shown to decrease anxiety-like behavior62 and increase social interaction time63, which has been used to model negative symptoms of the disorder. Conversely, activation of the pathway increases anxiety-like behaviors62 and decreases social interaction time63. The BLA has been implicated in schizophrenia64, therefore, it is likely that changes in this neural circuit may also contribute to the pathology of schizophrenia. However, schizophrenia is a complex disorder and the individual pathways examined do not exist in isolation. Further, each patient experiences a unique set of symptoms. Therefore, we and other believe that symptom clusters in schizophrenia result from disruptions in interconnected neural systems involving the pathways examined in the current experiments45.In the current experiments, we focused on pathways from the vHipp to the NAc and mPFC. However, schizophrenia is a heterogeneous disorder and the vHipp is not the only site of pathology. For example, structural and functional changes have also been observed in the thalamus of schizophrenia patientsA receptor in pyramidal cells can attenuate schizophrenia-like deficits in a rodent model. Further, we showed that discrete projections from the vHipp differentially mediate symptoms of the disorder. We believe that by better understanding the neuronal pathways associated with discrete dimensions of antipsychotic efficacy, novel therapeutics can be developed with improved efficacy at treating multiple symptom domains associated with schizophrenia.In conclusion, we have demonstrated that normalizing hippocampal activity using a gene therapy strategy to over-express the α5 subunit of the GABAad libitum unless specified below.All experiments were performed in accordance with the guidelines outlined in the USPH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio. Rats were maintained on a 12 h/12 h light/dark cycle, with food and water available 12. Male pups were weaned on postnatal day 21 and housed in groups of 3 until they were >12 weeks old, at which point rats were singly housed and used for behavioral or electrophysiological experiments. All experiments included pups from multiple litters.To model circuit level alterations and behavioral deficits associated with schizophrenia, timed pregnant female Sprague–Dawley rats were obtained from Harlan on gestational day 16. Methylazoxymethanol (22 mg/kg i.p.) or saline was administered on gestational day 17, a dose and time point that induces schizophrenia-like changes in behavior and neuronal activityA receptor under the control of the CAMKII promoter, lentiviral vectors were used. On postnatal days 40–45, animals were anesthetized using Fluriso  and placed in a stereotaxic apparatus. Bilateral cannula aimed at the vHipp , α5 virus  or the control virus  into each hemisphere . Animals were allowed to recover for >6 weeks before behavioral and electrophysiological experiments to allow maximal gene expression.To over-express the α1 or α5 subunit of the GABA47, AAV2 vectors driven by the CAMKII promoter were used. On postnatal days 40–45, animals were anesthetized (Fluriso), placed in a stereotaxic apparatus and bilateral cannula were used to inject the Gi virus (RAAV2-CAMKIIα-HA-hM4D(Gi)-IRES-mcitrine; 1.4 × 1012 vm/ml, 1.0 ul) or the control virus  into each vHipp .To over-express the designer receptor hM4D (Gi), a modified version of the human Gi-coupled muscarinic receptor 4 that inhibits neuronal activity in response to the exogenous ligand, clozapine-N-oxide70 or the mPFC . Brains were cut into horizontal 300 µm slices with a Vibratome . Slices contain the hippocampus were incubated in standard aCSF containing (in mM): 140 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, and 0.4 ascorbic acid  at room temperature for at least 1 h before recording commenced.It is well known that ectopic expression of receptors may not necessarily recapitulate the physiological role of that receptor in vivo. To better understand the consequence of a1 and a5 overexpression in vHipp pyramidal neurons, we employed whole-cell patch-clamp electrophysiology. Brains were removed and placed in ice-cold artificial cerebrospinal fluid (aCSF) containing (in mM): 261 sucrose, 2 KCl, 2 MgSO2, 10 HEPES, 1 EGTA, 4 NaCl, 2 Mg-ATP, 5 QX-314 . Tonic GABA currents and spontaneous IPSC activity were recorded in whole-cell configuration in voltage-clamp mode (Vhold = −70 mV) using an Axopatch 200B amplifier and pCLAMP software . Signals were filtered at 2 kHz, digitized 10 kHz , and saved on a computer for offline analysis. Recordings were made from neurons identified by the AAV-encoded fluorescent reporter GFP using an appropriate filter set. Recordings were performed with AMPA and NMDA receptors blocked with CNQX (10 µM) and DL-AP5 (50 µM), respectively. Tonic GABA current was measured as a reduction of holding current following bath application of picrotoxin (100 µM) for ~5 min. Amplitude and frequency of sIPSC activity were quantified using Clampfit software (v10.3) from ~3 min of stable baseline data recorded at least 5 min after achieving whole-cell configuration and prior to picrotoxin exposure.Patch-clamp recordings from hippocampal CA1 pyramidal neurons were performed with the aid of IR-DIC optics and a 16-bit EMCCD digital camera . Patch electrodes were pulled  from borosilicate glass capillaries and polished to a tip resistance of 3–5 MΩ. Electrodes were filled with a solution containing (in mM): 120 CsCl, 1 MgCl−1, i.p.), which does not appreciably affect dopamine system function65. Rats were then placed in a stereotaxic apparatus. Supplemental anesthesia was administered as necessary and a core body temperature of 37 °C was maintained. Extracellular glass microelectrodes were lowered into the VTA  and recorded using LabChart software . It should be noted that; while dopamine neurons projecting to different brain regions display distinct electrophysiological signatures67, the ability to physiologically identify dopamine neurons in vivo has been recently confirmed in a review by Ungless and Grace68. The following parameters of dopamine neuron activity were analyzed: (1) population activity, which was defined as the average number of spontaneously activity neurons recorded per electrode track, (2) basal firing rate, and (3) the proportion of action potentials occurring in bursts .To measure the activity of dopamine neurons in the ventral tegmental area (VTA), rats were anesthetized with 8% chloral hydrate (400 mg kg69) were recorded. The firing rate was analyzed using Labchart software.In order to measure putative pyramidal cell activity in the vHipp, we performed in vivo extracellular recordings as described above. Extracellular glass microelectrodes were lowered into the vHipp  and putative pyramidal neurons  that extended 1 mm past the end of the indwelling cannula were used to inject 300 uM  CNO into the NAc or mPFC.To measure cognitive flexibility, the AST was used. Rats were restricted to 12 g food/day for 7 days prior to testing. Using a cheerio reward, rats were trained to dig in pots defined by cues along two stimulus dimensions: the digging medium filling the pot and an odor  applied to the inner rim of the pot. During testing, the rat was taken through a series of stages, each requiring a different discrimination, with a criterion of six consecutive correct trials required to proceed to the next stage. The first stage was a simple discrimination (SD), with only one stimulus dimension (odor or medium) present. Odor (clove or nutmeg) was the initial relevant dimension  for half the animals, and medium (raffia or yarn) for the other half. The second stage was a compound discrimination (CD) in which the same discrimination was required and the second irrelevant dimension was introduced. The third stage was a reversal (R1) in which the same odors and media were used, and the same dimension remained relevant, but the negative cue from the previous stage became positive and the positive cue from the previous stage was now negative. Stage 4 was an intradimensional shift (ID), in which all new odors (cinnamon or rosemary) and media  were introduced but the same dimension remained relevant. After performing a second reversal (R2), the last stage was an extradimensional set-shift (ED). During ED, all new odors (citronella or thyme) and media (velvet or crepe paper) were presented and the previously irrelevant stimulus became relevant.Immunohistochemistry was used to confirm virus-mediated gene expression in vHipp neurons. Briefly, after behavioral and physiological experiments were performed, rats were transcardially perfused with saline, followed by 4% paraformaldehyde. Brains were post-fixed and cryoprotected in 10% sucrose in phosphate-buffered saline (PBS). For antigen retrieval, free-floating coronal sections from the vHipp (50 μm) were boiled in 10 mM citric acid, pH 6, for a total of 5 min. Sections were then washed in PBS, blocked , then incubated with chicken anti-GFP antibody  at 4 °C overnight. Sections were then incubated with AlexaFluor 488 goat anti-chicken secondary antibody (1:1000). To confirm pyramidal cell expression, a subset of sections were then incubated with mouse anti-CAMKII antibody  followed by AlexaFluor 594 goat anti-mouse (1:1000). Sections were mounted on slides and cover-slipped using Prolong gold anti-fade reagent. Sections were imaged using an Olympus IX81 Motorized Inverted microscope. The representative images were acquired using FV10-ASW software and enhanced using ImageJ.n is indicated in the figure legend. In the patch-clamp experiments, data were analyzed using a one-way ANOVA followed by the Holm–Sidak post-hoc test. For all other experiments, data were analyzed using a two-way ANOVA and Holm–Sidak post-hoc test. All tests were two tailed and significance was determined to at p < 0.05.Animals were randomly assigned to treatment groups. In all figures, data are shown as mean ± SEM and Further information on research design is available in the Peer Review FileReporting Summary


Klinefelter syndrome (KS) has a prevalence ranging from 85 to 250 per 100.000 newborn boys making it the most frequent sex chromosome aneuploidy in the general population. The molecular basis for the phenotypic traits and morbidity in KS are not clarified. We performed genome-wide DNA methylation profiling of leucocytes from peripheral blood samples from 67 KS patients, 67 male controls and 33 female controls, in addition to genome-wide RNA-sequencing profiling in a subset of 9 KS patients, 9 control males and 13 female controls. Characterization of the methylome as well as the transcriptome of both coding and non-coding genes identified a unique epigenetic and genetic landscape of both autosomal chromosomes as well as the X chromosome in KS. A subset of genes show significant correlation between methylation values and expression values. Gene set enrichment analysis of differentially methylated positions yielded terms associated with well-known comorbidities seen in KS. In addition, differentially expressed genes revealed enrichment for genes involved in the immune system, wnt-signaling pathway and neuron development. Based on our data we point towards new candidate genes, which may be implicated in the phenotype and further point towards non-coding genes, which may be involved in X chromosome inactivation in KS. Ethnic differences in the prevalence of KS may exist1. The presence of the additional X chromosome is associated with a number of health problems involving multiple organs and consequently are both morbidity and mortality significantly increased3. The increased morbidity seen in KS is due to an increased risk of developing physical diseases such as diabetes, metabolic syndrome, obesity, cardiovascular disease, infections, osteoporosis, as well as psychiatric diseases3. In addition, many patients with KS suffer from cognitive disabilities and behavioral problems. However, the degree of co-morbidity seen between KS patients display great heterogeneity, and as a consequence, diagnosis is often delayed4 and thereby also prevention and treatment of associated comorbidities.Klinefelter syndrome  has a prevalence ranging from 85 to 250 per 100.000 liveborn males making it the most frequent sex chromosome aneuploidy in the general populationSHOX5. It is localized in the pseudoautosomal region of the sex chromosomes and likely causes the tall stature in KS due to the presence of a third copy5. In cells with more than one X chromosome, one of the X chromosome is inactivated6 in order to avoid a potentially toxic double dose of X-linked genes. It has been hypothesized that some phenotypic features seen in KS may be explained by an overexpression of the 15% of X chromosomal genes escaping X chromosomal inactivation and of the 10% showing a variable cell-type specific expression9 due to a dosage effect equal to that seen in female, but different from what is normally seen in males. In addition, the hormonal imbalance with hypergonadotropic hypogonadism caused by gonadal dysfunction in KS10 has also been linked to some of the co-morbidities seen in KS, such as the increased prevalence of diabetes, obesity and metabolic syndrome, osteoporosis and cognitive disabilities. Hypogonadism alone, however, cannot explain the entire KS phenotype as hypogonadism often first develops during adolescent/adulthood11, where many of the co-morbidities are already present. The underlying mechanisms, linking the extra X chromosome to the clinical phenotype and the associated co-morbidities seen in KS thus remain unexplained.To date, the only gene associated with the KS phenotype is 14. These genome-wide alterations in DNA methylation may play a role in the biological mechanisms underlying the clinical KS phenotype by affecting chromatin structure and gene expression and thereby potentially be responsible for the development of phenotypical traits and diseases. Interestingly, alterations of the trancriptome in blood, brain tissue and testis tissue in KS20 have also been demonstrated, thereby supporting the hypothesis that sex chromosomes may regulate gene expression throughout the genome21. However, thus far only one study has investigated the potential association between alterations in the methylome and changes in the transcriptome in KS patients however this study were performed on brain tissue and included a single KS patient13. In addition, all of the above mentioned methylation studies either were performed on small KS cohorts or used arrays with a relative small number of CpG sites. Also, only one of the studies assessing RNA expression in KS have included a comprehensive analysis of non-coding RNA in addition to coding-RNA19.Recently, a few studies have provided evidence that KS may be associated with widespread changes in the methylome of both blood and brain tissueTo perform a comprehensive characterization of the methylome as well as the transcriptome in KS including both coding as well as non-coding genes and to further investigate the association between alteration in the methylome and changes in the transcriptome in KS patients, we performed DNA methylation profiling and total RNA-Seq transcription profiling in a cohort of KS patients and in female and male controls. Our aim was to assess the functional impact of epigenetic alterations on the transcriptome in KS, to enhance the understanding of molecular mechanism behind the observed phenotype and the increased risk of comorbidities in addition to describe the methylome and transcriptome of KS.Characterization of the methylome as well as the transcriptome of both coding and non-coding genes identified a unique epigenetic and genetic landscape in KS, with correlation analysis between the methylome and transcriptome revealing few genes with a direct association. Gene set enrichment analysis based on the methylation and expression analysis yielded terms associated with well-known comorbidities seen in KS as well as an affected immune system, wnt-signaling pathway and neuron development. Based on our data, we point towards several candidate genes, which may be implicated in the phenotype of KS and further point towards ncRNAs, which may be involved in X chromosome inactivation in KS and in the regulation of escape genes.FUNDC1, KDM5C, LOC100132831, MED14, SHROOM2, TAF7L, TFDP3 and XIST). Extending our analysis to the autosomal CpG sites, we identified 168 DMPs  between KS and male controls , 67 male controls  and 33 female controls  using the 450K-Illumina Infinium assay. CpG methylation at autosomal loci as well as at X chromosomal loci clearly differed between KS and controls, as reflected by the clear separation of the three groups in principal component analyses Fig. . Compari22. We therefore extended our methylation analysis to the regional level. We identified nine X chromosomal DMRs between KS and female controls, all located to genes   (i.e. methylation in groups of nearby positions) have been suggested to serve a functional role in gene transcriptional regulationWe explored the distribution of autosomal DMPs from the KS vs male controls, and KS vs female controls comparison in relation to CpG island status. Each CpG site of the 450 K Illumina array are annotated according to their relation to CpG islands. Six annotation categories exist. These include (1) within a CpG island, (2) north shore (within 2 kb upstream of an island), (3) south shore (within 2 kb downstream of an island), (4) north shelf (within 2–4 kb upstream of an island), (5) south shelf (within 2–4 kb downstream of an island) or 6) sea (none of the above-mentioned locations). We found a significant difference in the distribution of hypermethylated, but not hypomethylated DMPs compared to the distribution of all autosomal loci on the array, with hypermethylated DMPs enriched inside CpG islands and north shores Fig. . To furt23. Repetitive elements include transposons, which often target protein-coding genes, which can lead to genomic instability and thereby predispose to cancer and other diseases24. We therefore wanted to investigate if KS was associated with altered methylation level in these repetitive elements compared to male controls. Probes located to repetitive elements (n = 19465) were investigated and of these 202 were hypermethylated and 79 hypomethylated in KS compared to male controls (FWER > 0.05). Only two DMPs, both hypomethylated, reached a FWER > 0.05 and absolute delta-Beta value > 0.1  to genes and performed gene set enrichment. Gene-centric modular analysis revealed enrichment for terms related to diabetes, obesity, height, coronary and arterial disease, hypercholesterolemia, gingivitis and periodontitis, bone mineral density, cancer , connective tissue diseases, infection and inflammation and others  differentially expressed in both KS contrasts   Fig. , Table 1.3) Fig. , Table 19. Genes escaping X chromosomal inactivation have been suggested to influence the phenotype seen in KS, due to a dosage effect equal to that seen in female, but different from what is normally seen in males. Furthermore, genes located in the pseudoautosomal regions (PAR1 and PAR2) of the X and Y chromosomes have been proposed as candidate genes for the phenotypic traits seen in KS, due to the extra copy of these genes, compared to the normal two copies. We therefore annotated X chromosomal genes to escape (n = 133), inactivated (n = 177) or pseudoautosomal genes (n = 16)21. No clear clustering was seen between the three groups based on RNA-Seq data of neither escape, inactivated nor pseudoautosomal genes  was differentially expressed in both KS contrasts; however, upregulated compared to male controls and downregulated compared to female controls  a Y homolog exist. A decreased expression level was found in female controls compared to KS and male controls when investigating the expression of five of these 6 respective X-Y homologues . No expression data were available for TXLNGY. For inactivated genes, we identified three DEGs between KS and male controls, of which one gene, AMOT, was downregulated in both KS contrasts  was upregulated in both contrasts    between both KS contrasts  account for the remaining 40%nes Fig. . Howevernes Fig.  in contrnes Fig. , 797  Fig. , 36 (14 25) Fig. , and 52 25) Fig. . JPX wassts Fig. . No diffols Fig. .Figure 5We conducted correlation analysis of the 31 DEG between KS and male controls of which 16 were also differently expressed between KS and female controls, to investigate which DEGs correlated within the KS group. We found a positive correlation between IF144 vs. IF144L , IF144 vs. IFFIFIT1 , IF144 vs. RSAD2  and IF144L and HERC5 .BOLA1, DDX43, ZBTB44) as well as one X chromosomal gene (KDM5C) . For X chromosomal genes, the analysis was performed for the KS vs. female controls comparison (corresponding to three genes). We found a moderate correlation for three autosomal genes  overlapping between KS and male controls    . In addition, 5 of these genes  were found to be upregulated in Zitzmann et al.17, whereas only one (XIST) of these nine DEGs were upregulated according to Vawter et al.16, XIST were also found to be upregulated in Huang et al.20. Six other DEGs found in our study were also found to be upregulated in Zitzmann et al.17, . ZFX was also found to be upregulated in Vawter et al.16. In a murine study, DDX3X, KDM5C and EIF2S3 was upregulated in brain tissue from 41,XXY mouse compared to the 40,XY mouse29.In addition, only few studies have performed gene expression profiling in KS 30. These results could imply that a gain or loss of an X chromosome in humans cause epigenetic instability or alterations, which may be implicated in the phenotype seen in sex chromosome aneuploidies, through an effect on transcriptional or translational regulation. In support of this hypothesis, previous studies on cell lines found evidence that the presence of aberrant chromosome material may cause epigenomic instability by changing the regulation of transcription31.Our finding that KS was mainly associated with hypermethylation and to a lesser extent hypomethylation, are in agreement with earlier studiesSPEG, G3BP1, NSD1, ZNF497 and SHROOM2. However, based on the known functional roles of these genes, it is plausible that these methylation changes could be implicated in the phenotypic traits seen in KS such as delayed motor development33, decreased muscle strength35, cardiac abnormalities36, cognitive deficits37, and endothelial dysfunction38  were also upregulated in KS compared to male controls in the study by Zitzmann et al.17. In addition, Belling et al.15 also found EIF1AX, EIF2S3 and PRKX upregulated in KS compared to male controls. However, when we took into account the Y homolog of 5 of our 10 differentially expressed escape genes , no difference in expression values were seen between KS and male controls. The Y homologs were not taken into account in the studies by Belling et al.15. and Zitzmann et al.17. Thus, based on the rather limited available published studies performing gene expression profiling, the demonstrated altered expression of the TXLNG and EIF2S3 genes, likely contribute to the disease pathogenesis of KS. TXLNG may regulate bone mass density and is an interesting candidate gene of the decreased bone mass and the increased risk of developing osteoporosis in KS. EIF2S3 has been linked to X linked mental retardation, hypogonadism, obesity, microcephaly and epilepsy and among its related pathways are regulation of lipid metabolism and insulin. Further, it has been suggested to contribute to spermatogenesis. As such, these 2 genes may be implicated in several of the phenotypic traits seen in KS, such as osteoporosis34, dyslipidemia41, diabetes41, obesity41, decreased total brain volume42, hypogonadism and azoospermia10  in KS compared to male controls, with only SLC25A6 to be significantly upregulated in KS in agreement with Zitzmann et al.17 and Belling et al.15SLC25A6 is involved in calcium signaling pathway and metabolism. Interestingly, Zitzmann et al. found that higher expression of SLC25A6 was correlated with shorter QTc. Thus, overexpression of SLC25A6 may be part of the molecular mechanism behind short QTc interval in KS patients43  as well as between males and females (n = 29) which may indicate that the X inactivation is not equal to that seen in females, but is modified towards an expression pattern similar to that seen in males, which were further supported by our findings of only 3 differentially expressed inactivated genes between KS and males. One of the inactivated genes, AMOT was downregulated compared to both female controls and male controls. AMOT is expressed in endothelial cells of capillaries and plays a role in endothelial cell-cell junctions. AMOT could thus be involved in the endothelial dysfunction seen in KS38, which may be part of the pathogenesis underlying comorbidities in KS, such as hypertension, diabetes, atherosclerosis and erectile dysfunction. Interestingly, we found a significantly higher expression of JPX in KS compared to both male and female controls and a significantly higher expression of TSIX compared to male controls but not female controls. We speculated if these findings could be related to the X chromosome inactivation process in KS, however further studies are needed to investigate these hypothesis.In cells with more than one X chromosome, additional X chromosomes are inactivatedKDM5C, ZFX and EIF2S3)50, indicating that these non-coding genes could be involved in the regulation of the X chromosome inactivation and the expression of escape genes, which has to be further investigated in future studies.We found several non-coding genes to be differentially expressed in KS compared to both male controls and female controls, including many with unknown function. The biological implications of the aberrant expression of these non-coding genes remain to be elucidated. Interestingly, some of the differentially expressed X chromosomal non-coding genes were located in close proximity to the X chromosome inactivation center, or in close proximity to known escape genes  and 67 age-matched male controls  (mean age ± SD: 36.3 ± 9.71 years) in addition to 33 female controls female controls  (mean age ± SD: 45.0 ± 10.3 years) from a previous study of Turner syndrome . DNA methylation profiling was performed on peripheral blood samples from all participants. Gene expression profiling were performed on peripheral blood samples from a subset of participants including 9 KS (mean age ± SD: 36.0 ± 6.78 years), 9 male controls (mean age ± SD: 36.2 ± 7.14 years) and 13 female controls (mean age ± SD: 35.0 ± 7.34). All participants provided informed consent. The study was approved by The Danish Data Protection Agency and the local ethics committee .EDTA treated peripheral blood samples from participants were stored immediately until use at −80 °C. Genomic DNA was extracted using QIAmp® Mini Kit . For each sample, 1 μg of genomic DNA was bisulfite-converted using Zymo EZ DNA methylation Kit. The methylation level was measured using the Infinium® HumanMethylation450 Beadchip Kit  at Aros Applied Biotechnology A/S.minfi were used to analyze data52. Detection p-values were calculated to identify failed positions with a p-value cut-off >0.01. Probes that failed in more than 20% of the samples were removed (n = 183). Individual positions were removed at the specified cut-off. No samples had a proportion of failed probes exceeding 1% or a median intensity below 11. Raw data was normalized implementing the preprocessing defaults of Genome Studio® with background normalization and control normalization. Next, we applied subset-quantile-within-array-normalization correcting for technical differences between Infinium type I and II assay design allowing both within-array and between-sample normalization. Cross reactive probes , probes with SNPs documented in the C or G of the target , and probes on the sex chromosomes  were excluded, leaving 414,960 probes. Methylation values where calculated as M-values (logit[beta]) (Equation (1)53:et al.54.The R package Minfi’s estimateCellCounts was used for returning the relative proportions of CD4+ and CD8+ T-cells, natural killer cells, monocytes, granulocytes, and B-cells in each sample55.To account for differences in cell composition, Limma) allowing for missing values. Significance was evaluated using F-test. The sample variances were estimated using an empirical Bayes approach with shrinkage towards the means. A Bonferroni adjusted family wise error rate (FWER) below 0.05 was considered significant. The model was applied without and subsequently with adjustment for the estimated relative cell proportions  as well as age. Since small changes in M-values might be of spurious biological significance we added a delta M-value-threshold excluding all DMPs with a |delta-M-value| < 1. Chromosomal and gene-centric-region enrichment analysis was done applying hypergeometric testing to the KS vs. male controls comparison in order to identify if any chromosome or gene centric region was enriched for methylation changes.To identify positions where methylation is associated with the karyotype we fitted a linear model, which utilizes a generalized least squares model  < 0.05 with a |delta-M-value| > 0.1 was considered significant. Following FDR-correction, regions were agglomerated from groups of significant probes with a distance of less than 1000 base pairs to the next significant probe. Only DMRs with two or more probes are reported.DMRcate was used to identify autosomal as well as X chromosomal DMRsDMPs were annotated to the human genome version 19. DMPs common to both the KS vs. male controls and KS vs. female controls adjusted comparison with a FWER < 0.05 and absolute delta-M-value > 0.5. Enrichment of functional terms among associated genes were analysed with DAVID . A background list composed of genes corresponding to the 414,960 probes left for downstream analysis were entered. The database performs a biological module-centric analysis viewing functionally related genes together as a unit in order to identify the most overrepresented biological terms associated with a given gene list. The DAVID functional annotation clustering measures the relationship among the annotation terms on the basis of the degree of their co-association with genes within the entered gene list to cluster into functional annotation groups. Terms with Fisher’s exact p-value < 0.05 are reported.DMRs with a FWER < 0.05 and absolute delta-M-value > 0.1 were annotated using GREAT (The Genomic Regions Enrichment of Annotations Tool). DMRs were mapped to genes, assigned a regulatory domain extended 10 kb proximal and 3 kb downstream of the transcription start site with a distal extension of 150 kb. We included the curated Regulatory Domain option.Blood samples were drawn using RNApax gene tubes and placed 2 hours at room temperature, sequentially stored overnight at −21 degrees before storage at −80 degrees.Whole transcriptome, strand-specific RNA-Seq libraries were prepared from total-RNA using the Ribo-Zero Globin technology  for depletion of rRNA and globin mRNA followed by library preparation using the ScriptSeq technology . Depletion and library preparation were automated on a Sciclone NGS  liquid handling robot. The total-RNA (1.7 µg per sample) was subjected to Baseline-ZERO DNase prior to depletion. Total-RNA was purified using Agencourt RNAClean XP Beads before and after DNase treatment followed by on-chip electrophoresis on a LabChip GX and by UV measurements on a NanoQuant(Tecan). Cytoplasmic and mitochondrial rRNA as well as globin mRNA were removed from 400 ng DNAse treated total RNA using the Ribo-Zero Globin Gold Kit  following the manufacturer’s instructions and quality of the depleted RNA was estimated on a LabChip GX . Synthesis of directional RNA-Seq libraries were conducted using the ScriptSeq v2 kit  following the recommended procedure, and the qualities of the RNA-Seq libraries were estimated by on-chip electrophoresis  of a 1 µL sample. The DNA concentrations of the libraries were estimated using the KAPA Library Quantification Kit (Kapa Biosystems). The RNA-Seq libraries were multiplexed paired-end sequenced on an Illumina HiSeq. 2000 (100 + 6 + 100 bp) or on an Illumina NextSeq (75 + 6 + 75 bp).58. The gene expression were performed on two iterations, first on protein coding genes extracted from gencode v15 and non-coding transripts were extracted from gencode v19 and supplements from the mirbase, mitranscriptome, rfam, snornabase, tjumirna and trnascanse databases. HTSeq-count (union method) was applied to produce raw counts submitted for analysis in R using edgeR59. All non-informative features were removed. Filtering was done by removing features with less than one counts per million (cpm) in 9 samples leaving 11407 autosomale coding genes, 6380 non-coding autosomale RNAs, 424 X chromsomale coding genes, 231 non-coding X chromosomal RNAs for downstream analysis60. A generalized linear model was fitted yielding an overall p-value. Secondly, p-values and log fold changes (LogFC) were retrieved from the individual comparison of KS vs. male controls, KS vs. female controls and male controls vs. female controls. Genes from the comparison KS vs. male controls were submitted to DAVID for functional annotation pathways. Categories with a Fisher´s exact test p-value < 0.05 were retrieved. The FDR was controlled using the Benjamini-Hochberg´s procedure. Information on X-Y homologs was adapted from Bellot et al.21 and HGNC gene symbols and ensemble gene IDs were retrieved. Fragments Per Kilobase of Exon values (FPKM) of the X-Y pairs were calculated and summed. Differential expression was assessed with Kruskal-Wallis test and Mann-Whitney U-test. We used the gene annotation by Bellott et al.21.Paired de-multiplexed fastq files were generated using CASAVA software (Illumina) and initial quality control was performed using FastQC. Adapter trimming was conducted using the GATK ReadAdaptorTrimmer tool followed by mapping to the human genome (hg19) using Tophat61. DNA methylation data was assessed using the minfi52, DMRcate56 and Limma57 package, and RNAseq data using edgeR. Graphics were made using the basic R functions, ggbio, Gviz, DEseq60, DEXSeq and ggplot262. The package knitr was used for data documentation. Ideogram were created using Ideographica63.Statistical computations were performed using R 3.1.0  with Bioconductor 3.0https://ega-archive.org “The European Genome-phenome Archive of human data consented for biomedical research.Sequence data has been deposited at the European Genome-phenome Archive (EGA), which is hosted by the EBI and the CRG, under accession numbers EGAS00001002190 and EGAS00001002797. Further information about EGA can be found on Supplementary Information


DRD2 itself and its molecular effectors, have been implicated as key genetic predictors of response to treatments. Studies have also reported that genetic variation in genes coding for proteins that cross-talk with DRD2 at the molecular level, such as AKT1, GSK3B, Beta-catenin, and PPP2R2B are associated with response to antipsychotics. In this review we discuss the relative contribution to antipsychotic drug responsiveness of candidate genes and GWAS identified genes encoding proteins involved in dopamine responses. We also suggest that in addition of these older players, a deeper investigation of new GWAS identified schizophrenia risk genes such as FXR1 can provide new prospects that are not clearly engaged in dopamine function while being targeted by dopamine-associated signaling molecules. Overall, further examination of genes proximally or distally related to signaling mechanisms engaged by medications and associated with disease risk and/or treatment responsiveness may uncover an interface between genes involved in disease causation with those affecting disease remediation. Such a nexus would provide realistic targets for therapy and further the development of genetically personalized approaches for schizophrenia.Antipsychotic drugs targeting dopamine neurotransmission are still the principal mean of therapeutic intervention for schizophrenia. However, about one third of people do not respond to dopaminergic antipsychotics. Genome wide association studies (GWAS), have shown that multiple genetic factors play a role in schizophrenia pathophysiology. Most of these schizophrenia risk variants are not related to dopamine or antipsychotic drugs mechanism of action. Genetic factors have also been implicated in defining response to antipsychotic medication. In contrast to disease risk, variation of genes coding for molecular targets of antipsychotics have been associated with treatment response. Among genes implicated, those involved in dopamine signaling mediated by D2-class dopamine receptor, including  Whole genome association studies (GWAS) have identified several single nucleotide polymorphisms (SNPs) associated with enhanced risk for schizophrenia . These sAlternatively, several genetic factors have been shown to modulate the severity of schizophrenia symptoms and treatment responsiveness. Twins studies have demonstrated that, like pathophysiology and risk for schizophrenia, response to antipsychotic medication is a heritable trait , 5. Furtsubstantia nigra pars compacta to the caudate nucleus and the putamen in the striatum  Figure . This issα G-proteins and encoded by genes that are devoid of introns. D2-class dopamine receptors DRD2, DRD3, and DRD4, are encoded by genes that comprise introns and are generally coupled to Gi/oα G-proteins. D1-class receptors mediate post-synaptic responses to dopamine. In contrast D2-class receptors can both mediate post-synaptic responses and act as presynaptic auto-receptors to limit dopamine synthesis and release (DRD2 gene encodes two splice variants of the receptor. The long isoform (D2L) is mostly expressed on post-synaptic neurons while the short isoform (D2S) is preferentially expressed by pre-synaptic dopamine neurons  can be modulated by dopamine. Subunits of AMPA and NMDA ionotropic glutamate receptors  are also regulated by PKA downstream of dopamine receptors (Activation of D1-class receptors results in an increased production of the second messenger cyclic Adenosine Monophosphate (cAMP) by class 3 adenylyl cyclases (ADCY) . Activateceptors , 18. Fineceptors , 29.Qα G-proteins to regulate intracellular inositol and calcium signaling  and G-protein gated inwardly rectifying potassium channels   . Further2/KCNJ6) . This le2/KCNJ6) , 34. In 2/KCNJ6) , 36. The2/KCNJ6) . However2/KCNJ6) . The RNA2/KCNJ6)  that has as modulators of antipsychotic drug responsiveness and schizophrenia symptoms. In particular, genetics studies on schizophrenia endo-phenotypes, i.e., behavioral and/or neuroimaging phenotypes associated with the disorder, have suggested that variations in genes coding for this cluster of receptors, especially those coding for DRD2, DRD3, and DRD4, are associated with cognitive deficits and related brain pre-frontal cortex malfunction, which are typical phenotypes of schizophrenia. Candidate gene investigations have identified allelic variation in SNPs of functional relevance to D2, D3, and D4 receptors to be associated with response to antipsychotic medication  is a missense variant determining a Glu-to-Lys substitution at position 713 of DRD2 gene and associated with the gene mRNA stability and expression (DRD2 gene and determining a Ser-to-Cys substitution at position 311 of DRD2 gene (Ser311Cys) has been shown to alter the physiology and function of DRD2 receptor  has been associated with altered DRD3 dopamine affinity and density in some brain areas , is a 48 bp Variable Number Tandem Repeat (48.bp VNTR) falling within the third exon of the gene and resulting in a different length of the third cytoplasmatic loop of DRD4 protein. Importantly, the longer repeat version of the protein has been associated with reduced clozapine binding, thus suggesting the 48 bp VNTR to be an interesting candidate polymorphism to study as predictor of clinical response to this antipsychotic. However, while some studies have reported an effect of rs4646984 on responsiveness to various antipsychotics including clozapine  as compared to the Val/Val condition. A large number of studies have investigated the genetic association between rs4680 and the diagnosis of schizophrenia with mixed results—for a meta-analysis see:  has been described . Such an approach has confirmed the polygenic nature of risk for schizophrenia since it has identified more than one hundred genetic variants associated with the disorder at genome wide statistical level of significance   . Neverth < 10−8) .DRD2, have been associated with schizophrenia by GWAS, even though, most findings have implicated different variants from those detected by candidate gene approaches  , a multiDRD2 gene. This same study identified SNPs associated with olanzapine and risperidone response in Ankyrin Repeat and Sterile Alpha Motif Domain-Containing Protein 1B (ANKS1B) and in the Contactin-Associated protein-Like 5 genes (CNTNAP5) which, are not directly connected with dopamine signaling, but are involved in interneuron communication.The first GWAS  found a GRIA4, codes for AMPA 4 glutamate receptor, that may impact on dopamine neurotransmission by mediation of glutamate signaling. In fact, evidence suggests that aberrant glutamatergic function may alter dopamine system function in psychotic disorders (GRIN2A) or indirectly related to dopaminergic system . The remaining two genes, CACNA1C and HCN1, respectively coding for LTCC Cav 1.2 and Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, have also been implicated in dopamine signaling modulation  and is caused by mutations in FMR1 gene, which is highly expressed in neurons and plays a crucial role in regulating synaptic plasticity –119. BotFMR1 are not associated to schizophrenia as defined by a GWAS studies of common genetic risk variants  has shown it to be in linkage disequilibrium with splicing quantitative trait loci (sQTL) SNP (rs1805564) identified in postmortem human dorsolateral pre-frontal cortex (DLPFC). Altered splicing in the locus of exon 15 is associated and functionally changed in presence of the rs34796896 schizophrenia risk allele , which is necessary for efficient shuttling between cytoplasm and nucleoli. This shuttling was demonstrated in Cos-7 cell line for the isoform  or FMR1 . Lately, or FMR1 . Interes or FMR1 , 115. ThFXR1 may also contribute to DRD2 and GSK3β mediated signaling . Indeed,FXR1 (rs496250) and GSK3B (rs12630592) genes  genes . In heal2) genes . Finally2) genes . EvidencGenetic investigations of risk factors for schizophrenia and determinants of drug responsiveness revealed a very multi-genic landscape for both indications. This suggests that schizophrenia arises from a combination of multiple genetic and socio-environmental hits occurring during development. This is also in line with the variable profile of drug responsiveness observed at the clinical level. This picture can be discouraging as several risk factors may only participate to disease at a pre-onset stage or, contribute to ubiquitous functions across different organs, thus preventing their practical or ethical use as therapeutic targets. Nonetheless, the overlap of schizophrenia risk and genes affecting responsiveness to existing drugs also points toward a nexus of biological mechanisms engaged by medication and those contributing to disease etiology. This supports the idea that disease remediation can intersect with disease causation and help compensate for developmental insults, even during adulthood. The prevalence of genes involved in dopamine transmission among those associated with treatment responsiveness is flagrant. However, dopamine is an “old player” in schizophrenia therapy and the prevalence of dopamine-associated genes probably results from a bias induced by the mechanism of action of therapeutic agents. The further examination of genes that are more distal to dopamine signaling and are nonetheless associated with schizophrenia risk and drug responsiveness may provide a new roster of “prospects” that can be realistically targeted for therapies whether alone, or in conjunction with older dopamine-targeting therapeutics. Furthermore, the integrated investigation of new and old variants associated both to genetic risk and treatment responsiveness can provides the bases for the development of personalized treatment protocols for schizophrenia.All authors wrote the manuscript. JB also provided funds for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Langerin+CD8+ DC exhibit a high ability to take up apoptotic/dying cells, and therefore they are essential to prime and shape CD8+ T cell responses. Next to the induction of immunity toward blood-borne pathogens, i.e., viruses, these DC are important for the regulation of tolerance toward cell-associated self-antigens. The ontogeny and differentiation pathways of CD8+CD103+ DC should be further explored to better understand the immunological role of these cells as a prerequisite of their therapeutic application.Dendritic cells (DC) fulfill an essential sentinel function within the immune system, acting at the interface of innate and adaptive immunity. The DC family, both in mouse and man, shows high functional heterogeneity in order to orchestrate immune responses toward the immense variety of pathogens and other immunological threats. In this review, we focus on the Langerin Dendritic cells (DC) link pathogen sensing and activation of innate immunity to the initiation of (primary) adaptive immune responses. For the latter, DC function as professional antigen (Ag)-presenting phagocytes that orchestrate the priming and polarization of naïve T cells. Importantly, next to stimulating protective immunity following infection, cancer or vaccination, DC are also crucial for the maintenance of immunological (self-) tolerance.intCD45RA+Ly6C+ cells). These pDC are poor in Ag-presentation but play a crucial role as first-line defense against viral infections and are involved in anti-tumor responses as well , marginal metallophilic macrophages (MMM) and marginal zone B cells (MZB), thereby initiating immune responses against systemic pathogens –13 Figu. Moreove+CD11b− cDC1 subset, and a CD11b+ cDC2 subpopulation that can be further divided into (ii) CD4+CD8− DC and (iii) CD4−CD8− double-negative DC subsets. To date, unsupervised phenotypic analysis, for example using (single cell) RNA sequencing and high-dimensional flow cytometry or mass cytometry, has added a large number of additional subpopulation-specific markers, confirming the existence of heterogeneity (DC subsets) within both cDC1 and cDC2 subpopulations  a CD8αα-expressing CD8ulations . All of ulations .+ cDC1 co-express the C-type lectin receptors DEC205 (CD205) and Langerin (CD207)  . InitialALS only , 17–20, 7 . Initio the WP , 21–23. o the WP –28. Thiso the WP   via TGFβ  cells via CD1d presentation of glycolipid Ag  are, nevertheless, mainly attributed to the cDC1 subpopulation as a whole. However, differential expression of DEC205 and CXdulation , 41  are preferentially involved in MHCII-restricted Ag presentation and TH2 priming  and CD4−CD8− double-negative cDC2 are often collectively referred to as CD8− cDC2 -DC progenitors and monocytes, it is still under debate whether these splenic ESAMlow cDC originate from circulating monocytes or not , although this does not result in clearly defined homogenous populations , which mD8− cDC2 , 49–52, D8− cDC2 , 53. Fors or not , 54. Moss or not , 55.− cDC2 reside in the MZ and bridging channels of the spleen  depends on Notch2  express Langerin and are functionally distinct  in the epidermis and skin-draining lymph nodes , 62. Howdistinct –66. In athe lung –70. Amonlocation . Althougpulation , 72–75.+CD8+ cDC1 are primarily localized in the MZ, just internal to the F4/80+ RP Mϕ, interspersed with MZM and forming a ring around the CD169+ MMM  or glycolipid antigen α-galactosylceramide  stimulation are similar to Langerin+CD8+ cDC1  cell ligand α-Gal-Cer also resulted in a fast decline of splenic Langerin+CD8+ cDC1 numbers, which peaked after 15–24 h. However, Langerin−CD8+ cDC1 numbers remained unchanged, suggesting that activation does not convert Langerin+ into Langerin− cDC1 but that Langerin+CD8+ cDC1 are rather sensitive to activation-induced cell death  in the BM, but the exact developmental pathways of different cDC lineages remain controversial and difficult to elucidate , 81–86 . It is g numbers , 93–95.+CD8+ cDC1-specific transcriptional program has been identified. Moreover, both Langerin+CD8+ and Langerin−CD8+ cDC1 express similar levels of the cDC1-associated transcription factors Irf8, Id2, Nfil3, and Batf3  could functionally restore the CX3CR1−Langerin−CD8+ cDC1 compartment in Batf3 KO mice, while these mice still lack the majority of splenic Langerin+CD8+ cDC1  Langerin+CD8+ subset, and that the differentiation into mature Langerin+CD8+ DC depends on Batf3 and yet undefined conditions  in the splenic MZ exhibit a high phagocytic capacity for dying / apoptotic cells, suggesting that the MZ is essential for the initiation of immune self-tolerance -mediated Ag targeting to the recognition receptors DEC205 and Clec9a resulted in efficient cross-presentation and CTL priming . As this pro-apoptotic molecule induces apoptosis when diverted into the cytoplasm, cyt c specifically depleted cells that possess cytosolic export mechanisms required for cross-presentation  by Langerin+CD8+ cDC1 remains unknown. However, DEC205 and Langerin Ag-targeting experiments revealed that, in contrast to Langerin+ migratory skin cDC, Langerin+CD8+ cDC1 in the spleen were inefficient in generating TREGin vivo , where mainly Mϕ in the peripheral RP actively remove these senescent RBC (erythrophagocytosis) . However+ cDC .+CD8+ cDC1 subset is predominantly involved in clearance and cross-presentation of circulating apoptotic cells, but not, or only minimally, in MHCII presentation and subsequent CD4+ T cell priming in responses to the same Ag. Again, this does not answer whether the ability to cross-present depends on a certain maturation stage characterized by the expression of Langerin, or whether Langerin+CD8+ and Langerin−CD8+ cDC1 are two functionally discrete cDC1 subsets, and this should be examined.In summary, the Langerin+ cDC1 subset, or whether the receptor actually exhibits functional properties. In LC, Langerin is associated with the formation of Birbeck granules. However, these structures are absent in Langerin+CD8+ cDC1, and their formation cannot be induced by stimulation with anti-Langerin monoclonal Ab  to reinforce vaccination efficiency is of great interest. Currently, many protein-targeting strategies utilize DEC205 and Clec9a receptors, but this will result in targeting of additional cell types as their expression is not cDC restricted . In contrategies .+CD8+ cDC1 actually exist? And if so, what will be their relevance and how much of the murine knowledge is translatable to the human system? At first glance, similar to mice, the human DC network can be divided into multiple phenotypically and functionally distinct cDC1 and cDC2 resident and migratory DC subpopulations , suggesting that some cDC2 are included in the Langerin+ cDC fraction as well  may facilitate the specific properties of, exclusively, the Langerin+CD8+ cDC1 subset. Therefore, the elucidation of the relationship between Langerin+CD8+ cDC1 and the MZ, including the determination of factors supporting the unique properties of Langerin+CD8+ cDC1, may be of particular interest. The combination of high-dimensional techniques and unbiased analysis has already revealed distinct differentiation stages and/or subpopulations of human cDC1 and cDC2  immune functions attributed to the splenic cDC1 subpopulation appear to be exerted by the Langerinand cDC2 , 126, anAll authors listed have made a substantial, direct and intellectual contribution to writing this review article, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.