Edible mycelium bioengineered for enhanced nutritional value and sensory appeal using a modular synthetic biology toolkit.

Filamentous fungi are critical in the transition to a more sustainable food system. While genetic modification of these organisms has promise for enhancing the nutritional value, sensory appeal, and scalability of fungal foods, genetic tools and demonstrated use cases for bioengineered food production by edible strains are lacking. Here, we develop a modular synthetic biology toolkit for Aspergillus oryzae, an edible fungus used in fermented foods, protein production, and meat alternatives. Our toolkit includes a CRISPR-Cas9 method for gene integration, neutral loci, and tunable promoters. We use these tools to elevate intracellular levels of the nutraceutical ergothioneine and the flavor-and color molecule heme in the edible biomass. The strain overproducing heme is red in color and is readily formulated into imitation meat patties with minimal processing. These findings highlight the promise of synthetic biology to enhance fungal foods and provide useful genetic tools for applications in food production and beyond.

Combinatorial gene therapy for epilepsy: Gene sequence positioning and AAV serotype influence expression and inhibitory effect on seizures.

Gene therapy with AAV vectors carrying genes for neuropeptide Y and its receptor Y2 has been shown to inhibit seizures in multiple animal models of epilepsy. It is however unknown how the AAV serotype or the sequence order of these two transgenes in the expression cassette affects the actual parenchymal gene expression levels and the seizure-suppressant efficacy. To address these questions, we compared three viral vector serotypes (AAV1, AAV2 and AAV8) and two transgene sequence orders (NPY-IRES-Y2 and Y2-IRES-NPY) in a rat model of acutely induced seizures. Wistar male rats were injected bilaterally with viral vectors and 3 weeks later acute seizures were induced by a subcutaneous injection of kainate. The latency until 1st motor seizure, time spent in motor seizure and latency to status epilepticus were measured to evaluate the seizure-suppressing efficacy of these vectors compared to an empty cassette control vector. Based on the results, the effect of the AAV1-NPY-IRES-Y2 vector was further investigated by in vitro electrophysiology, and its ability to achieve transgene overexpression in resected human hippocampal tissue was evaluated. The AAV1-NPY-IRES-Y2 proved to be better to any other serotype or gene sequence considering both transgene expression and ability to suppress induced seizures in rats. The vector also demonstrated transgene-induced decrease of glutamate release from excitatory neuron terminals and significantly increased both NPY and Y2 expression in resected human hippocampal tissue from patients with drug-resistant temporal lobe epilepsy. These results validate the feasibility of NPY/Y2 receptor gene therapy as a therapeutic opportunity in focal epilepsies.

Neuroglobin deficiency increases seizure susceptibility but does not affect basal behavior in mice.

Neuroglobin (Ngb) is found in the neurones of several different brain areas and is known to bind oxygen and other gaseous molecules and reactive oxygen species (ROS) in vitro, but it does not seem to act as a respiratory molecule for neurones. Using male and female Ngb-knockout (KO) mice, we addressed the role of Ngb in neuronal brain activity using behavioral tests but found no differences in general behaviors, memory processes, and anxiety-/depression-like behaviors. Oxidative stress and ROS play key roles in epileptogenesis, and oxidative injury produced by an excessive production of free radicals is involved in the initiation and progression of epilepsy. The ROS binding properties led us to hypothesize that lack of Ngb could affect central coping with excitatory stimuli. We consequently explored whether exposure to the excitatory molecule kainate (KA) would increase severity of seizures in mice lacking Ngb. We found that the duration and severity of seizures were increased, while the latency time to develop seizures was shortened in Ngb-KO compared to wildtype adult female mice. Consistently, c-fos expression after KA was significantly increased in Ngb-KO mice in the amygdala and piriform cortex, regions rich in Ngb and known to be centrally involved in seizure generation. Moreover, the measured c-fos expression levels were correlated with seizure susceptibility. With these new findings combined with previous studies we propose that Ngb could constitute an intrinsic defense mechanism against neuronal hyperexcitability and oxidative stress by buffering of ROS in amygdala and other Ngb-containing brain regions.

Serum sclerostin levels in osteoporotic fracture patients.

PURPOSE: Sclerostin inhibits bone formation and stimulates bone resorption. Previous studies found a positive association between bone density and serum sclerostin, but literature on sclerostin levels in osteoporotic fracture patients is scarce. The aim of the present study was to compare the serum sclerostin levels in osteoporotic and non-osteoporotic fracture patients and to assess the correlation of the sclerostin levels with bone mineral density and vitamin D status. METHODS: In this cross-sectional study, we included patients over 50 years, with an extremity fracture after low-energy trauma treated between 2012 and 2018, with biobank samples and available bone density measurements by Dual X-ray Absorption. Osteoporosis was diagnosed according the World Health Organisation criteria. Vitamin D deficiency was defined as a 25(OH)D concentration < 30 nmol/L. After defrosting biobank samples, serum sclerostin was measured using the human SOST (sclerostin) enzyme-linked immunosorbent assay kit. We prespecified a subgroup analysis including only female patients. RESULTS: 179 patients were included of whom 139(78%) were female. In 46 patients (25.7%), osteoporosis was diagnosed. Bone mineral density was positively associated with sclerostin levels (r = 0.17, p = 0.026) and patients with osteoporosis had a significantly lower serum sclerostin compared to non-osteoporotic fracture patients (mean 41.9 pmol/L vs 48.1 pmol/L; p = 0.03). This difference remained significant after correction for potential confounders. Similar results were found in the subgroup of female patients. No association between serum sclerostin and vitamin D deficiency was found. CONCLUSION: Osteoporotic fracture patients had lower levels of sclerostin than non-osteoporotic fracture patients. Future research should focus on the use of sclerostin as biomarker for osteoporosis in fracture patients.

No effect of fixation type on early and late mortality after total knee arthroplasty: a Dutch arthroplasty register study.

PURPOSE: Postoperative mortality is commonly reported as outcome measurement after total knee arthroplasty (TKA). Mortality might be influenced by multiple factors including cementation of the prosthesis. Until now, the influence of cementation on early and late mortality after TKA is unknown. The aim of the present study was to determine the effect of fixation on early and late mortality after primary TKA. METHODS: All patients in the Dutch Arthroplasty Register (LROI) with a primary TKA for osteoarthritis were eligible for inclusion. Data collected from 2007 to 2014 with follow-up until January 2020 were used. Survival analysis was performed by using Kaplan-Meier and Cox survival analysis to determine the mortality rate according to fixation. Adjustments were made for age at time of surgery, gender, American Society of Anaesthesiologists class, and year of surgery. RESULTS: In total 108,687 TKA were included for analysis, which comprised 95,857 cemented, 6,140 cementless and 6,690 hybrid TKA. The early and late mortality rate in cemented TKA was statistically not different compared to cementless or hybrid TKA at 30 days, 31-90 days, 91 days-1 year and 1-5 years. The hazard ratio at 30 days was 1.05 (CI 0.49-2.25) for hybrid fixation, and 1.46 (CI 0.74-2.90) for cementless fixation compared to cemented fixation. The 1-5 years hazard ratio was 1.06 (CI 0.96-1.17) and 0.97 (CI 0.87-1.08), respectively. CONCLUSION: Based on register data, method of fixation does not influence early mortality after primary TKA. This suggests that there is no preferred fixation technique for primary TKA based on the mortality rates. LEVEL OF EVIDENCE: IV.

Comparable mid-term revision rates of primary cemented and cementless total knee arthroplasties in 201,211 cases in the Dutch Arthroplasty Register (2007-2017).

PURPOSE: Long-term failure of total knee arthroplasty (TKA) is mostly due to loosening of the prosthesis. In this study, the short- and mid-term revision rates of cemented vs cementless TKAs were investigated. Comparable short- and mid-term survival rates of both fixation methods were expected. METHODS: Data on all cemented and cementless TKAs performed between 2007 and 2017 were retrieved from the Dutch Arthroplasty Register. The cumulative crude incidence of revision of cemented and cementless TKA was calculated. Death was considered a competing risk. Revision rates were compared using multivariable Cox proportional hazard regression analysis. The associations between fixation method and type of revision or reason for revision were tested using logistic regression analyses. RESULTS: In total, 190,651 (94.8%) cemented and 10,560 (5.3%) cementless TKAs were evaluated. Both groups had comparable case characteristics. Cemented TKAs were inserted more often in cases with previous knee surgery compared to cementless TKAs (32% vs 27%). The cumulative incidence of revision after 9 years was 5.5% (CI 5.3-5.6%) for cemented and 5.8% (CI 5.2-6.4%) for cementless TKAs (p = 0.2). Cementless TKAs were more often revised due to loosening of the tibial (27% vs 18%; p < 0.001) or the femoral component (7% vs 5%; p = 0.005) than cemented TKAs. Cemented TKAs were more often revised due to infection (17% vs 9%; p = 0.004) than cementless TKAs. CONCLUSION: In conclusion, cemented and cementless TKAs have comparable short- and mid-term revision rates based on a nationwide register study. LEVEL OF EVIDENCE: III.

The soluble neurexin-1ß ectodomain causes calcium influx and augments dendritic outgrowth and synaptic transmission.

Classically, neurexins are thought to mediate synaptic connections through trans interactions with a number of different postsynaptic partners. Neurexins are cleaved by metalloproteases in an activity-dependent manner, releasing the soluble extracellular domain. Here, we report that in both immature (before synaptogenesis) and mature (after synaptogenesis) hippocampal neurons, the soluble neurexin-1ß ectodomain triggers acute Ca2+-influx at the dendritic/postsynaptic side. In both cases, neuroligin-1 expression was required. In immature neurons, calcium influx required N-type calcium channels and stimulated dendritic outgrowth and neuronal survival. In mature glutamatergic neurons the neurexin-1ß ectodomain stimulated calcium influx through NMDA-receptors, which increased presynaptic release probability. In contrast, prolonged exposure to the ectodomain led to inhibition of synaptic transmission. This secondary inhibition was activity- and neuroligin-1 dependent and caused by a reduction in the readily-releasable pool of vesicles. A synthetic peptide modeled after the neurexin-1ß:neuroligin-1 interaction site reproduced the cellular effects of the neurexin-1ß ectodomain. Collectively, our findings demonstrate that the soluble neurexin ectodomain stimulates growth of neurons and exerts acute and chronic effects on trans-synaptic signaling involved in setting synaptic strength.

GnRH agonist and hCG (dual trigger) versus hCG trigger for final follicular maturation: a double-blinded, randomized controlled study.

STUDY QUESTION: Does co-administration of GnRH agonist and Human chorionic gonadotropin (hCG; dual trigger) in IVF cycles improve the number of mature oocytes and pregnancy outcome compared to hCG alone? SUMMARY ANSWER: Using the dual trigger for final follicular maturation increases the number of oocytes, mature oocytes and number of blastocysts (total and top-quality) compared to triggering with hCG alone. WHAT IS KNOWN ALREADY: hCG is used at the end of controlled ovarian hyperstimulation as a surrogate LH surge to induce final oocyte maturation. Recently, based on retrospective studies, the co-administration of GnRH agonist and hCG for final oocyte maturation (dual trigger) has been suggested to improve IVF outcome and pregnancy rates. STUDY DESIGN, SIZE, DURATION: A single center, randomized controlled, double-blinded clinical trial between May 2016 and June 2018 analyzed by intention to treat (ITT). PARTICIPANTS/MATERIALS, SETTINGS, METHODS: One hundred and fifty-five normal responder patients were randomized either to receive hCG or dual trigger for final oocyte maturation. Data on patients age, BMI, AMH, number of oocytes retrieved, number of metaphase 2 (MII) oocytes, zygotes and blastocysts, clinical pregnancy rate and live birth rate were assessed and compared between the dual trigger group and the hCG group. We performed a planned interim analysis after the recruitment of 50% of the patients. Based on the totality of outcomes at the interim analysis we decided to discontinue further recruitment. MAIN RESULTS AND THE ROLE OF CHANCE: One hundred and fifty-five patients were included in the study. The age (36 years versus 35.3 years P = NS), BMI (24 kg/m2 versus 23.7 kg/m2) and the AMH (20.1 pmol/l versus 22.4 pmol/l) were comparable between the two groups. Based on ITT analysis, the number of eggs retrieved (11.1 versus 13.4, P = 0.002), the MII oocytes (8.6 versus 10.3, P = 0.009), total number of blastocysts (2.9 versus 3.9, P = 0.01) and top-quality blastocysts transferred (44.7% versus 64.9%; P = 0.003) were significantly higher in the dual trigger group compared to the hCG group. The clinical pregnancy rate (24.3% versus 46.1%, OR 2.65 (1.43-1.93), P = 0.009) and the live birth rate per transfer (22% versus 36.2%, OR= 1.98 (1.05-3.75), P = 0.03) were significantly higher in the dual trigger group compared to the hCG group. LIMITATIONS, REASONS FOR CAUTION: None. WIDER IMPLICATIONS OF THE FINDINGS: The enhanced response observed with the dual trigger might lead to better IVF outcomes were it used more widely. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by TRIO Fertility. There are no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov identifier: NCT02703584. DATE OF TRIAL REGISTRATION: March 2016. DATE OF FIRST PATIENT'S ENROLLMENT: May 2016.

A novel approach for eliciting adolescent MDR-TB treatment tolerability: qualitative data from South Africa.

SETTING: Treatment tolerability among adolescents diagnosed with multidrug-resistant tuberculosis (MDR-TB) is underexplored. We present qualitative study data from adolescents participating in an observational cohort in the Western Cape, South Africa.OBJECTIVE: To elicit adolescent experiences of MDR-TB diagnosis and treatment with qualitative body-mapping activities and discussions.DESIGN: Adolescents in an observational MDR-TB cohort received routine toxicity and audiology screenings from clinicians. We enrolled eight participants (age 10-16 years) to participate in additional body-mapping activities and in-depth interviews. A thematic deductive analysis was conducted. We present a comparison of the clinical assessments and qualitative discussions.RESULTS: Adolescent participants reported few adverse effects on standard toxicity and audiology reports. Only nausea and vomiting were reported in >10% of cases, all of which were grade 1 (causing no/minimal interference) adverse effects (AEs). However, when comparing toxicity reports with qualitative body-mapping activities and interviews, we found previously unreported AEs (neurosensory alteration, neuromuscular weakness, pain); underestimated severity of AEs (nausea, itching); and missed psychosocial symptoms (signs of depression).CONCLUSION: Adolescents receiving treatment for MDR-TB experienced treatment-related AEs that were not reported during routine clinical assessments. Psychosocial experiences of adolescents are not taken into account. More research is needed to understand the experiences of this vulnerable group. We recommend that drug safety monitoring be adapted to include more creative and patient-driven reporting mechanisms for vulnerable groups, including children.

Deterministic generation of a two-dimensional cluster state.

Measurement-based quantum computation offers exponential computational speed-up through simple measurements on a large entangled cluster state. We propose and demonstrate a scalable scheme for the generation of photonic cluster states suitable for universal measurement-based quantum computation. We exploit temporal multiplexing of squeezed light modes, delay loops, and beam-splitter transformations to deterministically generate a cylindrical cluster state with a two-dimensional (2D) topological structure as required for universal quantum information processing. The generated state consists of more than 30,000 entangled modes arranged in a cylindrical lattice with 24 modes on the circumference, defining the input register, and a length of 1250 modes, defining the computation depth. Our demonstrated source of two-dimensional cluster states can be combined with quantum error correction to enable fault-tolerant quantum computation.

Disease Modification by Combinatorial Single Vector Gene Therapy: A Preclinical Translational Study in Epilepsy.

Gene therapy has been suggested as a plausible novel approach to achieve seizure control in patients with focal epilepsy that do not adequately respond to pharmacological treatment. We investigated the seizure-suppressant potential of combinatorial neuropeptide Y and Y2 receptor single vector gene therapy based on adeno-associated virus serotype 1 (AAV1) in rats. First, a dose-response study in the systemic kainate-induced acute seizure model was performed, whereby the 1012 genomic particles (gp)/mL titer of the vector was selected as an optimal concentration. Second, an efficacy study was performed in the intrahippocampal kainate chronic model of spontaneous recurrent seizures (SRSs), designed to reflect a likely clinical scenario, with magnetic resonance image (MRI)-guided focal unilateral administration of the vector in the hippocampus during the chronic stage of the disease. The efficacy study demonstrated a favorable outcome of the gene therapy, with a 31% responder rate (more than 50% reduction in SRS frequency) and 13% seizure-freedom rate, whereas no such effects were observed in the control animals. The inter-SRS and SRS cluster intervals were also significantly prolonged in the treated group compared to controls. In addition, the SRS duration was significantly reduced in the treated group but not in the controls. This study establishes the SRS-suppressant ability of the single vector combinatorial neuropeptide Y/Y2 receptor gene therapy in a clinically relevant chronic model of epilepsy.

Sustained overexpression of neuropeptide S in the amygdala reduces anxiety-like behavior in rats.

Neuropeptide S (NPS) has shown anxiolytic-like effects in rodents after acute administration, but its long-term effects remain unknown. Gene therapy enables the targeted delivery of DNA to cell nuclei, and recombinant adeno-associated viral (rAAV) vectors have been identified as suitable tools for stable overexpression. Thus, to explore the effects of long-term expression of NPS, the present study examined anxiety- and depressive-like effects after rAAV-mediated NPS overexpression in the rat amygdala. Compared to rats injected with an empty control vector (rAAV-Empty), rAAV-NPS treatment was associated with reduced anxiety-like behavior in the elevated plus maze and light-dark box, but did not affect depressive-like behavior in the forced swim test. Importantly, rAAV-NPS did not cause confounding effects on locomotion or bodyweight as opposed to currently used anxiolytic drugs. Immunohistochemical stainings revealed NPS-positive cells in the central and basolateral region of the amygdala in rAAV-NPS but not rAAV-Empty rats, indicating successful transduction. Our study provides novel evidence for sustained anxiolytic-like properties of NPS by transgenic overexpression. These data suggest that rAAV-NPS application deserves further attention as a potential treatment strategy for anxiety in humans.

Decreased spontaneous activity in AMPK α2 muscle specific kinase dead mice is not caused by changes in brain dopamine metabolism.

It is well known that physical activity has several health benefits, yet many people do not exercise. Dopamine levels in the striatum of the brain are thought to be important for the motivation to exercise. Conversely, we hypothesized that muscle quality can affect the motivation to exercise through alterations of the brain dopamine levels specifically in the striatal region. To test this hypothesis, transgenic mice overexpressing an inactivatable dominant negative α2 AMPK construct (AMPK α2 KD) in muscles and littermate wildtype (WT) mice were tested. AMPK α2 KD mice have impaired running capacity and display reduced voluntary wheel running activity. Striatal content of dopamine and its metabolites were measured under basal physiological conditions and after cocaine-induced dopamine efflux from the ventral striatum by in vivo microdialysis. Moreover, cocaine-induced locomotor activity was tested in an open field test. Furthermore, we investigated maximal running capacity and voluntary running over a period of 19days. AMPK α2 KD mice ran 30% less in daily distance compared to WT. Furthermore, AMPK α2 KD mice showed significantly decreased locomotor activity in the open field test compared to WT when treated with saline or cocaine, respectively, but the increase induced by cocaine was similar in AMPK α2 KD and WT mice. The efflux of dopamine in ventral striatum after cocaine treatment increased similarly by 2.5-fold in the two genotypes, and basal levels of dopamine and its metabolites DOPAC and HVA were also similar between genotypes. These findings show that decreased AMPK activity in muscle leads to decreased voluntary activity which is not due to secondary abnormalities in dopamine levels in the ventral striatum or sensitivity to cocaine. Thus, decreased voluntary activity in AMPK muscle deficient mice is most likely unrelated to regulation of brain dopamine content and metabolism.

Translational approach for gene therapy in epilepsy: Model system and unilateral overexpression of neuropeptide Y and Y2 receptors.

Although novel treatment strategies based on the gene therapy approach for epilepsy has been encouraging, there is still a gap in demonstrating a proof-of-concept in a clinically relevant animal model and study design. In the present study, a conceptually novel framework reflecting a plausible clinical trial for gene therapy of temporal lobe epilepsy was explored: We investigated (i) whether the post intrahippocampal kainate-induced status epilepticus (SE) model of chronic epilepsy in rats could be clinically relevant; and (ii) whether a translationally designed neuropeptide Y (NPY)/Y2 receptor-based gene therapy approach targeting only the seizure-generating focus unilaterally can decrease seizure frequency in this chronic model of epilepsy. Our data suggest that the intrahippocampal kainate model resembles the disease development of human chronic mesial temporal lobe epilepsy (mTLE): (i) spontaneous seizures originate in the sclerotic hippocampus; (ii) only a part of the animals develops chronic epilepsy; (iii) animals show largely variable seizure frequency that (iv) tends to progressively increase over time. Despite significant hippocampal degeneration caused by the kainate injection, the use of MRI allowed targeting the recombinant adeno-associated viral (rAAV) vectors encoding NPY and Y2 receptor genes to the remaining dorsal and ventral hippocampal areas ipsilateral to the kainate injection. Continuous video-EEG monitoring demonstrated not only prevention of the progressive increase in seizure frequency in rAAV-NPY/Y2 treated animals as compared to the controls, but even 45% decrease of seizure frequency in 80% of the epileptic animals. This translationally designed study in a clinically relevant model of epilepsy suggests that simultaneous overexpression of NPY and Y2 receptors unilaterally in the seizure focus is a relevant and promising approach that can be further validated in more extensive preclinical studies to develop a future treatment strategy for severe, often pharmacoresistant focal epilepsy cases that cannot be offered alternative therapeutic options.

Activation of Neuropeptide Y Receptors Modulates Retinal Ganglion Cell Physiology and Exerts Neuroprotective Actions In Vitro.

Neuropeptide Y (NPY) is expressed in mammalian retina but the location and potential modulatory effects of NPY receptor activation remain largely unknown. Retinal ganglion cell (RGC) death is a hallmark of several retinal degenerative diseases, particularly glaucoma. Using purified RGCs and ex vivo rat retinal preparations, we have measured RGC intracellular free calcium concentration ([Ca2+]i) and RGC spiking activity, respectively. We found that NPY attenuated the increase in the [Ca2+]i triggered by glutamate mainly via Y1 receptor activation. Moreover, (Leu31, Pro34)-NPY, a Y1/Y5 receptor agonist, increased the initial burst response of OFF-type RGCs, although no effect was observed on RGC spontaneous spiking activity. The Y1 receptor activation was also able to directly modulate RGC responses by attenuating the NMDA-induced increase in RGC spiking activity. These results suggest that Y1 receptor activation, at the level of inner or outer plexiform layers, leads to modulation of RGC receptive field properties. Using in vitro cultures of rat retinal explants exposed to NMDA, we found that NPY pretreatment prevented NMDA-induced cell death. However, in an animal model of retinal ischemia-reperfusion injury, pretreatment with NPY or (Leu31, Pro34)-NPY was not able to prevent apoptosis or rescue RGCs. In conclusion, we found modulatory effects of NPY application that for the first time were detected at the level of RGCs. However, further studies are needed to evaluate whether NPY neuroprotective actions detected in retinal explants can be translated into animal models of retinal degenerative diseases.

Differential Effect of Neuropeptides on Excitatory Synaptic Transmission in Human Epileptic Hippocampus.

Development of novel disease-modifying treatment strategies for neurological disorders, which at present have no cure, represents a major challenge for today's neurology. Translation of findings from animal models to humans represents an unresolved gap in most of the preclinical studies. Gene therapy is an evolving innovative approach that may prove useful for clinical applications. In animal models of temporal lobe epilepsy (TLE), gene therapy treatments based on viral vectors encoding NPY or galanin have been shown to effectively suppress seizures. However, how this translates to human TLE remains unknown. A unique possibility to validate these animal studies is provided by a surgical therapeutic approach, whereby resected epileptic tissue from temporal lobes of pharmacoresistant patients are available for neurophysiological studies in vitro. To test whether NPY and galanin have antiepileptic actions in human epileptic tissue as well, we applied these neuropeptides directly to human hippocampal slices in vitro. NPY strongly decreased stimulation-induced EPSPs in dentate gyrus and CA1 (up to 30 and 55%, respectively) via Y2 receptors, while galanin had no significant effect. Receptor autoradiographic binding revealed the presence of both NPY and galanin receptors, while functional receptor binding was only detected for NPY, suggesting that galanin receptor signaling may be impaired. These results underline the importance of validating findings from animal studies in human brain tissue, and advocate for NPY as a more appropriate candidate than galanin for future gene therapy trials in pharmacoresistant TLE patients.

Neuropeptide Y-stimulated [(35) S]GTPγs functional binding is reduced in the hippocampus after kainate-induced seizures in mice.

Kainate-induced seizures constitute a model of temporal lobe epilepsy where prominent changes are observed in the hippocampal neuropeptide Y (NPY) system. However, little is known about the functional state and signal transduction of the NPY receptor population resulting from kainate exposure. Thus, in this study, we explored functional NPY receptor activity in the mouse hippocampus and neocortex after kainate-induced seizures using NPY-stimulated [(35) S]GTPγS binding. Moreover, we also studied levels of [(125) I]-peptide YY (PYY) binding and NPY, Y1, Y2, and Y5 receptor mRNA in these kainate-treated mice. Functional NPY binding was unchanged up to 12 h post-kainate, but decreased significantly in all hippocampal regions after 24 h and 1 week. Similarly, a decrease in [(125) I]-PYY binding was found in the dentate gyrus (DG) 1 week post-kainate. However, at 2 h, 6 h, and 12 h, [(125) I]-PYY binding was increased in all regions, and in the CA1 also at 24 h post-kainate. NPY mRNA levels were prominently increased in hippocampal regions, reaching maximum at 12 and 24 h. Y1 and Y5 mRNA levels were lowered in the DG at 24 and 2 h, respectively, while Y2 mRNA levels were elevated at 24 h in the DG and CA3. This study confirms rat kainate studies by showing pronounced adaptive changes in the mouse hippocampus both with regard to NPY synthesis and NPY receptor synthesis and binding, which may contribute to regulating neuronal seizure susceptibility after kainate. However, the potential seizure-suppressant effects of increased NPY gene expression at late time points post-kainate could be attenuated by the novel finding of reduced NPY-receptor G-protein activation.

Combined gene overexpression of neuropeptide Y and its receptor Y5 in the hippocampus suppresses seizures.

We recently demonstrated that recombinant adeno-associated viral vector-induced hippocampal overexpression of neuropeptide Y receptor, Y2, exerts a seizure-suppressant effect in kindling and kainate-induced models of epilepsy in rats. Interestingly, additional overexpression of neuropeptide Y in the hippocampus strengthened the seizure-suppressant effect of transgene Y2 receptors. Here we show for the first time that another neuropeptide Y receptor, Y5, can also be overexpressed in the hippocampus. However, unlike Y2 receptor overexpression, transgene Y5 receptors in the hippocampus had no effect on kainate-induced motor seizures in rats. However, combined overexpression of Y5 receptors and neuropeptide Y exerted prominent suppression of seizures. This seizure-suppressant effect of combination gene therapy with Y5 receptors and neuropeptide Y was significantly stronger as compared to neuropeptide Y overexpression alone. These results suggest that overexpression of Y5 receptors in combination with neuropeptide Y could be an alternative approach for more effective suppression of hippocampal seizures.

Adeno-associated viral vector-induced overexpression of neuropeptide Y Y2 receptors in the hippocampus suppresses seizures.

Gene therapy using recombinant adeno-associated viral vectors overexpressing neuropeptide Y in the hippocampus exerts seizure-suppressant effects in rodent epilepsy models and is currently considered for clinical application in patients with intractable mesial temporal lobe epilepsy. Seizure suppression by neuropeptide Y in the hippocampus is predominantly mediated by Y2 receptors, which, together with neuropeptide Y, are upregulated after seizures as a compensatory mechanism. To explore whether such upregulation could prevent seizures, we overexpressed Y2 receptors in the hippocampus using recombinant adeno-associated viral vectors. In two temporal lobe epilepsy models, electrical kindling and kainate-induced seizures, vector-based transduction of Y2 receptor complementary DNA in the hippocampus of adult rats exerted seizure-suppressant effects. Simultaneous overexpression of Y2 and neuropeptide Y had a more pronounced seizure-suppressant effect. These results demonstrate that overexpression of Y2 receptors (alone or in combination with neuropeptide Y) could be an alternative strategy for epilepsy treatment.