Another Use for a Proven Drug: Experimental Evidence for the Potential of Artemisinin and Its Derivatives to Treat Alzheimer's Disease.

Plant-derived multitarget compounds may represent a promising therapeutic strategy for multifactorial diseases, such as Alzheimer's disease (AD). Artemisinin and its derivatives were indicated to beneficially modulate various aspects of AD pathology in different AD animal models through the regulation of a wide range of different cellular processes, such as energy homeostasis, apoptosis, proliferation and inflammatory pathways. In this review, we aimed to provide an up-to-date overview of the experimental evidence documenting the neuroprotective activities of artemi-sinins to underscore the potential of these already-approved drugs for treating AD also in humans and propose their consideration for carefully designed clinical trials. In particular, the benefits to the main pathological hallmarks and events in the pathological cascade throughout AD development in different animal models of AD are summarized. Moreover, dose- and context-dependent effects of artemisinins are noted.

Primary cilia are critical for tracheoesophageal septation.

INTRODUCTION: Primary cilia play pivotal roles in the patterning and morphogenesis of a wide variety of organs during mammalian development. Here we examined murine foregut septation in the cobblestone mutant, a hypomorphic allele of the gene encoding the intraflagellar transport protein IFT88, a protein essential for normal cilia function. RESULTS: We reveal a crucial role for primary cilia in foregut division, since their dramatic decrease in cilia in both the foregut endoderm and mesenchyme of mutant embryos resulted in a proximal tracheoesophageal septation defects and in the formation of distal tracheo(broncho)esophageal fistulae similar to the most common congenital tracheoesophageal malformations in humans. Interestingly, the dorsoventral patterning determining the dorsal digestive and the ventral respiratory endoderm remained intact, whereas Hedgehog signaling was aberrantly activated. CONCLUSIONS: Our results demonstrate the cobblestone mutant to represent one of the very few mouse models that display both correct endodermal dorsoventral specification but defective compartmentalization of the proximal foregut. It stands exemplary for a tracheoesophageal ciliopathy, offering the possibility to elucidate the molecular mechanisms how primary cilia orchestrate the septation process. The plethora of malformations observed in the cobblestone embryo allow for a deeper insight into a putative link between primary cilia and human VATER/VACTERL syndromes.

Loss of Extrasynaptic Inhibitory Glycine Receptors in the Hippocampus of an AD Mouse Model Is Restored by Treatment with Artesunate.

Alzheimer's disease (AD) is characterized by synaptic failure and neuronal loss. Recently, we demonstrated that artemisinins restored the levels of key proteins of inhibitory GABAergic synapses in the hippocampus of APP/PS1 mice, a model of cerebral amyloidosis. In the present study, we analyzed the protein levels and subcellular localization of α2 and α3 subunits of GlyRs, indicated as the most abundant receptor subtypes in the mature hippocampus, in early and late stages of AD pathogenesis, and upon treatment with two different doses of artesunate (ARS). Immunofluorescence microscopy and Western blot analysis demonstrated that the protein levels of both α2 and α3 GlyRs are considerably reduced in the CA1 and the dentate gyrus of 12-month-old APP/PS1 mice when compared to WT mice. Notably, treatment with low-dose ARS affected GlyR expression in a subunit-specific way; the protein levels of α3 GlyR subunits were rescued to about WT levels, whereas that of α2 GlyRs were not affected significantly. Moreover, double labeling with a presynaptic marker indicated that the changes in GlyR α3 expression levels primarily involve extracellular GlyRs. Correspondingly, low concentrations of artesunate (≤1 µM) also increased the extrasynaptic GlyR cluster density in hAPPswe-transfected primary hippocampal neurons, whereas the number of GlyR clusters overlapping presynaptic VIAAT immunoreactivities remained unchanged. Thus, here we provide evidence that the protein levels and subcellular localization of α2 and α3 subunits of GlyRs show regional and temporal alterations in the hippocampus of APP/PS1 mice that can be modulated by the application of artesunate.

Strengths and Weaknesses of Non-enhanced and Contrast-enhanced Cadaver Computed Tomography Scans in the Teaching of Gross Anatomy in an Integrated Curriculum.

Cadaver-specific postmortem computed tomography (PMCT) has become an integral part in anatomy teaching at several universities. Recently, the feasibility of contrast-enhanced (CE)-PMCT has been demonstrated. The purpose of this study was to identify particular strengths and weaknesses of both non-enhanced and contrast-enhanced PMCT compared to conventional cadaver dissection. First, the students' perception of the learning effectiveness of the three different modalities have been assessed using a 34-item survey (five-point Likert scale) covering all anatomy course modules. Results were compared using the nonparametric Friedman Test. Second, the most frequent artifacts in cadaver CT scans, were systematically analyzed in 122 PMCT and 31 CE-PMCT data sets to quantify method-related limitations and characteristics. Perfusion quality was assessed in 57 vascular segments (38 arterial and 19 venous). The survey was answered by n = 257/320 (80.3%) students. Increased learning benefits of PMCT/ CE-PMCT compared to cadaver dissection were found in osteology (2/3 categories, P < 0.001), head and neck (2/5 categories, P < 0.01), and brain anatomy (3/3 categories, P < 0.01). Contrast-enhanced-PMCT was perceived particularly useful in learning vascular anatomy (10/10 categories, P < 0.01). Cadaver dissection received significantly higher scores compared to PMCT and CE-PMCT in all categories of the abdomen and thorax (7/7 categories, P < 0.001), as well as the majority of muscular anatomy (5/6 categories, P < 0.001). Frequent postmortem artifacts (total n = 28, native-phase n = 21, contrast injection-related n = 7) were identified and assessed. The results of this work contribute to the understanding of the value of integrating cadaver-specific PMCT in anatomy teaching.

Artemisinin-treatment in pre-symptomatic APP-PS1 mice increases gephyrin phosphorylation at Ser270: a modification regulating postsynaptic GABAAR density.

Artemisinins, a group of plant-derived sesquiterpene lactones, are efficient antimalarial agents. They also share anti-inflammatory and anti-viral activities and were considered for treatment of neurodegenerative disorders like Alzheimer's disease (AD). Additionally, artemisinins bind to gephyrin, the multifunctional scaffold of GABAergic synapses, and modulate inhibitory neurotransmission in vitro. We previously reported an increased expression of gephyrin and GABAA receptors in early pre-symptomatic stages of an AD mouse model (APP-PS1) and in parallel enhanced CDK5-dependent phosphorylation of gephyrin at S270. Here, we studied the effects of artemisinin on gephyrin in the brain of young APP-PS1 mice. We detected an additional increase of gephyrin protein level, elevated gephyrin phosphorylation at Ser270, and an increased amount of GABAAR-γ2 subunits after artemisinin-treatment. Interestingly, the CDK5 activator p35 was also upregulated. Moreover, we demonstrate decreased density of postsynaptic gephyrin and GABAAR-γ2 immunoreactivities in cultured hippocampal neurons expressing gephyrin with alanine mutations at two CDK5 phosphorylation sites. In addition, the activity-dependent modulation of synaptic protein density was abolished in neurons expressing gephyrin lacking one or both of these phosphorylation sites. Thus, our results reveal that artemisinin modulates expression as well as phosphorylation of gephyrin at sites that might have important impact on GABAergic synapses in AD.

Good healing potential of patellar chondral defects after all-arthroscopic autologous chondrocyte implantation with spheroids: a second-look arthroscopic assessment.

PURPOSE: To report second-look arthroscopic assessment after all-arthroscopic autologous chondrocyte implantation (ACI) for articular cartilage defects at the patella. METHODS: A second-look arthroscopy after all-arthroscopic ACI using chondrospheres® (ACT3D) was performed in 30 patients with 30 full-thickness retropatellar cartilage defects. The mean time from ACI to second-look arthroscopy was 14.9 ± 16.3 (6-71) months. The quality of cartilage regeneration was evaluated by the International Cartilage-Repair Score (ICRS)-Cartilage Repair Assessment (CRA). RESULTS: Eleven lesions (36.7%) were classified as CRA grade I (normal) and 19 lesions (63.3%) as grade II (nearly normal). Concerning the degree of defect repair, 25 lesions (83.3%) were repaired up to the height of the surrounding articular retropatellar cartilage. Five lesions (16.7%) showed 75% repair of defect depth. The border zone was completely integrated into the surrounding articular cartilage shoulder in 28 lesions (93.3%) and demarcated within 1 mm in 2 lesions (6.7%). Macroscopically and by probing, 12 lesions (40%) had intact smooth surface, 17 lesions (56.7%) had fibrillated surface and 1 lesion (3.3%) had small, scattered fissures. A negative correlation was found between the overall repair assessment score and the defect size (r2 = - 0.430, p = 0.046) and between integration into border zone and defect size (r2 = - 0.340, p = 0.045). A positive correlation was found between macroscopic appearance and age (r2 = + 0.384, p = 0.036). CONCLUSIONS: All-arthroscopic ACI using chondrospheres® (ACT3D) for full-thickness retropatellar articular cartilage defects proved to be reproducible and reliable. The advantage of the procedure is that it is minimal invasive. Arthroscopic second-look demonstrated a high grade of normal or nearly normal cartilage regeneration. Although statistically significant differences were not observed, larger defect size and younger age may compromise the result of overall repair. LEVEL OF EVIDENCE: III.

Nucleolar stress controls mutant Huntington toxicity and monitors Huntington's disease progression.

Transcriptional and cellular-stress surveillance deficits are hallmarks of Huntington's disease (HD), a fatal autosomal-dominant neurodegenerative disorder caused by a pathological expansion of CAG repeats in the Huntingtin (HTT) gene. The nucleolus, a dynamic nuclear biomolecular condensate and the site of ribosomal RNA (rRNA) transcription, is implicated in the cellular stress response and in protein quality control. While the exact pathomechanisms of HD are still unclear, the impact of nucleolar dysfunction on HD pathophysiology in vivo remains elusive. Here we identified aberrant maturation of rRNA and decreased translational rate in association with human mutant Huntingtin (mHTT) expression. The protein nucleophosmin 1 (NPM1), important for nucleolar integrity and rRNA maturation, loses its prominent nucleolar localization. Genetic disruption of nucleolar integrity in vulnerable striatal neurons of the R6/2 HD mouse model decreases the distribution of mHTT in a disperse state in the nucleus, exacerbating motor deficits. We confirmed NPM1 delocalization in the gradually progressing zQ175 knock-in HD mouse model: in the striatum at a presymptomatic stage and in the skeletal muscle at an early symptomatic stage. In Huntington's patient skeletal muscle biopsies, we found a selective redistribution of NPM1, similar to that in the zQ175 model. Taken together, our study demonstrates that nucleolar integrity regulates the formation of mHTT inclusions in vivo, and identifies NPM1 as a novel, readily detectable peripheral histopathological marker of HD progression.

Targeted Ablation of Primary Cilia in Differentiated Dopaminergic Neurons Reduces Striatal Dopamine and Responsiveness to Metabolic Stress.

Primary cilia (PC) are microtubule-based protrusions of the cell membrane transducing molecular signals during brain development. Here, we report that PC are required for maintenance of Substantia nigra (SN) dopaminergic (DA) neurons highly vulnerable in Parkinson's disease (PD). Targeted blockage of ciliogenesis in differentiated DA neurons impaired striato-nigral integrity in adult mice. The relative number of SN DA neurons displaying a typical auto-inhibition of spontaneous activity in response to dopamine was elevated under control metabolic conditions, but not under metabolic stress. Strikingly, in the absence of PC, the remaining SN DA neurons were less vulnerable to the PD neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin (MPTP). Our data indicate conserved PC-dependent neuroadaptive responses to DA lesions in the striatum. Moreover, PC control the integrity and dopamine response of a subtype of SN DA neurons. These results reinforce the critical role of PC as sensors of metabolic stress in PD and other disorders of the dopamine system.

Artesunate restores the levels of inhibitory synapse proteins and reduces amyloid-ß and C-terminal fragments (CTFs) of the amyloid precursor protein in an AD-mouse model.

Alzheimer's disease (AD) is the most frequent form of dementia, characterized histopathologically by the formation of amyloid plaques and neurofibrillary tangles in the brain. Amyloid ß-peptide (Aß) is a major component of amyloid plaques and is released together with carboxy-terminal fragments (CTFs) from the amyloid precursor protein (APP) through proteolytic cleavage, thought to contribute to synapse dysfunction and loss along the progression of AD. Artemisinins, primarily antimalarial drugs, reduce neuroinflammation and improve cognitive capabilities in mouse models of AD. Furthermore, artemisinins were demonstrated to target gephyrin, the main scaffold protein of inhibitory synapses and modulate GABAergic neurotransmission in vitro. Previously, we reported a robust decrease of inhibitory synapse proteins in the hippocampus of 12-month-old double transgenic APP-PS1 mice which overexpress in addition to the Swedish mutated form of the human APP a mutated presenilin 1 (PS1) gene and are characterized by a high plaque load at this age. Here, we provide in vivo evidence that treating these mice with artemisinin or its semisynthetic derivative artesunate in two different doses (10 mg/kg and 100 mg/kg), these compounds affect differently inhibitory synapse components, amyloid plaque load and APP-processing. Immunofluorescence microscopy demonstrated the rescue of gephyrin and γ2-GABAA-receptor protein levels in the brain of treated mice with both, artemisinin and artesunate, most efficiently with a low dose of artesunate. Remarkably, artemisinin reduced only in low dose the amyloid plaque load correlating with lower levels of mutated human APP (hAPPswe) whereas artesunate treatment in both doses resulted in significantly lower plaque numbers. Correspondingly, the level of APP-cleavage products, specifically the amount of CTFs in hippocampus homogenates was reduced significantly only by artesunate, in line with the findings in hAPPswe expressing cultured hippocampal neurons evidencing a concentration-dependent inhibition of CTF-release by artesunate already in the nanomolar range. Thus, our data support artemisinins as neuroprotective multi-target drugs, exhibiting a potent anti-amyloidogenic activity and reinforcing key proteins of inhibitory synapses.

Binding of gephyrin to microtubules is regulated by its phosphorylation at Ser270.

Gephyrin is a multifunctional scaffolding protein anchoring glycine- and subtypes of GABA type A- receptors at inhibitory postsynaptic membrane specializations by binding to the microtubule (MT) and/or the actin cytoskeleton. However, the conditions under which gephyrin can bind to MTs and its regulation are currently unknown. Here, we demonstrate that during the purification of MTs from rat brain by sedimentation of polymerized tubulin using high-speed centrifugation a fraction of gephyrin was bound to MTs, whereas gephyrin phosphorylated at the CDK5-dependent site Ser270 was detached from MTs and remained in the soluble protein fraction. Moreover, after collybistin fostered phosphorylation at Ser270 the binding of a recombinant gephyrin to MTs was strongly reduced in co-sedimentation assays. Correspondingly, upon substitution of wild-type gephyrin with recombinant gephyrin carrying alanine mutations at putative CDK5 phosphorylation sites the binding of gephyrin to MTs was increased. Furthermore, the analysis of cultured HEK293T and U2OS cells by immunofluorescence-microscopy disclosed a dispersed and punctuated endogenous gephyrin immunoreactivity co-localizing with MTs which was evidently not phosphorylated at Ser270. Thus, our study provides additional evidence for the binding of gephyrin to MTs in brain tissue and in in vitro cell systems. More importantly, our findings indicate that gephyrin-MT binding is restricted to a specific gephyrin fraction and depicts phosphorylation of gephyrin as a regulatory mechanism of this process by showing that soluble gephyrin detached from MTs can be detected specifically with the mAb7a antibody, which recognizes the Ser270 phosphorylated- version of gephyrin.

Influence of syndesmotic injuries and posterior malleolar ankle fractures on fibula position in the ankle joint: a cadaveric study.

PURPOSE: The aim of this study was to identify to what extent a dissection of the syndesmosis and an avulsed posterior edge of the tibia can change the tibiofibular diastasis and fibular rotation. METHODS: Three-dimensional scans with a mobile C-arm of 22 cadaver legs were taken of the intact fibula, after dissection of the anterior part of the syndesmosis and the interosseous membrane, osteotomy of the posterior malleolus, and osteosynthesis. The tibiofibular diastasis as well as the angle of fibular rotation was identified in the four steps and the means compared to each other using a t test for paired samples. RESULTS: The distinction between the intact fibula vs. the osteotomy of the posterior tibia was 0.082 ± 0.332 mm for the tibiofibular distance in the incisura tibiofibularis (p 0.261) and 0.046 ± 0.486 degrees for the angle of the fibular rotation (p 0.665). CONCLUSION: Neither the dissection of the syndesmosis nor the osteotomy of the posterior malleolus significantly influenced the position of the fibula in the incisura tibiofibularis in the cadaveric model. However, in the nonweight-bearing situation, a lesion of the syndesmotic complex might not be evident in intraoperative three-dimensional imaging.

Amyloid-ß Fosters p35/CDK5 Signaling Contributing to Changes of Inhibitory Synapses in Early Stages of Cerebral Amyloidosis.

Early changes in inhibitory synapse connectivities are thought to contribute to the excitation/inhibition imbalance preceding neurodegeneration in Alzheimer's disease (AD). Recently, we reported a robust increase in the level of different key-proteins of inhibitory synapses in hippocampal subregions of pre-symptomatic APPswe-PS1 mice, a model of cerebral amyloidosis. Besides increased inhibitory synaptic clusters on parvalbumin-positive projections in CA1 and CA3, we observed impaired communication between these two hippocampal areas of young APP-PS1 mice. Interestingly, the phosphorylation of gephyrin, a major organizer of inhibitory synapses, was also increased. Here, we demonstrate that the protein levels of CDK5, a kinase involved in the phosphorylation of gephyrin, and its regulatory protein p35 are also significantly increased in hippocampal subregions of young APP-PS1 mice. Consistently, the expression of hAPP-swe in cultured hippocampal neurons resulted in higher p35-protein levels, indicating a possible molecular link between increased Aß-production and the elevated p35/CDK5 levels seen in vivo. Further, a shRNA mediated downregulation of p35-expression in hippocampal neurons correlated with a decrease in gephyrin phosphorylation and in a reduced density of synaptic γ2-GABAA-receptor clusters. These findings, together with the detection of gephyrin colocalization with CDK5 and p35 by immunostaining and proximity ligation experiments in vivo and in vitro, are supporting our hypothesis that Aß has a profound impact on inhibitory network properties, likely mediated at least in part by p35/CDK5 signaling. This further underscores the impact of altered inhibitory synaptic transmission in AD.

Using Nonexpert Online Reports to Enhance Expert Knowledge About Causes of Death in Dental Offices Reported in Scientific Publications: Qualitative and Quantitative Content Analysis and Search Engine Analysis.

BACKGROUND: Fatalities rarely occur in dental offices. Implications for clinicians may be deduced from scientific publications and internet reports about deaths in dental offices. OBJECTIVE: Data involving deaths in dental facilities were analyzed using Google as well as the PubMed database. By comparing both sources, we examined how internet data may enhance knowledge about deaths in dental offices obtained from scientific medical publications, which causes of death are published online, and how associated life-threatening emergencies may be prevented. METHODS: To retrieve relevant information, we searched Google for country-specific incidents of death in dental practices using the following keywords: "death at the dentist," "death in dental practice," and "dying at the dentist." For PubMed searches, the following keywords were used: "dentistry and mortality," "death and dental treatment," "dentistry and fatal outcome," and "death and dentistry." Deaths associated with dental treatment in a dental facility, attributable causes of death, and documented ages of the deceased were included in our analysis. Deaths occurring in maxillofacial surgery or pre-existing diseases involved in the death (eg, cancer and abscesses) were excluded. A total of 128 cases from online publications and 71 cases from PubMed publications that met the inclusion criteria were analyzed using chi-square statistics after exclusion of duplicates. RESULTS: The comparison between the fatalities from internet (n=117) and PubMed (n=71) publications revealed that more casualties affecting minors appeared online than in PubMed literature (online 68/117, 58.1%; PubMed 20/71, 28%; P<.001). In PubMed articles, 10 fatalities in patients older than 70 years of age were described, while online sources published 5 fatalities (P=.02). Most deaths, both from internet publications and PubMed literature, could be assigned to the category anesthesia, medication, or sedation (online 80/117, 68.4%; PubMed 25/71, 35%; P<.001). Deaths assigned to the categories infection and cardiovascular system appeared more often in the PubMed literature (infection: online 10/117, 8.5%; PubMed 15/71, 21%; P=.01; cardiovascular system: online 5/117, 4.3%; PubMed 15/71, 21%; P<.001). Furthermore, sedative drugs were involved in a larger proportion of fatal incidents listed online compared to in PubMed (online 41/117, 35.0%; PubMed: 14/71, 20%, P=.03). In the United States, more deaths occurred under sedation (44/96, 46%) compared to those in the other countries (Germany and Austria 1/17, 6%, P=.002; United Kingdom 1/14, 7%, P=.006). CONCLUSIONS: Online and PubMed databases may increase awareness of life-threatening risks for patients during dental treatment. Negative aspects of anesthesia and sedation, as well as the number of deaths of young patients, were underestimated when reviewing PubMed literature only. Medical history of patients, medication dosages, and vital function monitoring are significant issues for practitioners. A high-impact finding from online reports was the underestimation of risks when performing sedation and even general anesthesia. Detailed knowledge of the definition and understanding of deep sedation and general anesthesia by dentists is of major concern. By avoiding potentially hazardous procedures, such as sedation-aided treatments performed solely by dentists, the risk of treatment-induced life-threatening emergencies may be reduced.

Influence of ankle joint position on angles and distances of the ankle mortise using intraoperative cone beam CT: A cadaveric study.

BACKGROUND: The precise anatomical reduction of the ankle mortise is crucial for the clinical outcome in unstable syndesmotic injuries. Intraoperative cone beam computed tomography (CT), in addition to two-dimensional fluoroscopy, provides detailed information about the reduction and implant placement. The aim of this study was to analyze the influence of the joint position on the fibula position in the incisural notch and to determine the inter- and intraindividual anatomical differences in the intact ankle joints. METHODS: A total of 20 fresh-frozen lower legs disarticulated in the knee joint of 10 individuals were included. The measurements were performed using a cone beam CT. The distances and angles were measured in the standard imaging planes. The mean values of distances and angles were compared during the different joint positions: 10° dorsiflexion, 0° neutral position and 20° plantar flexion. RESULTS: The influence of the joint position was on average as follows: The anterior tibiofibular distance was 3.68 mm in 10° dorsiflexion, 3.66 mm (0° neutral position) and 3.59 mm (20° plantar flexion). The posterior tibiofibular distance measured 7.82mm, 7.76mm and 7.82mm. The rotation of the fibula measured ten millimeters proximal the joint line was 1.2°, 1.3° and 1.05°. The fibular rotation determined 4mm was 9.3°, 9.4° and 9.4°. On average, the following intraindividual variations were observed: superior tibiotalar clear space of 0.27mm and 0.15mm medial; and anterior tibiofibular distance of 0.42mm, 0.38mm posterior and 0.24mm in the incisural notch. The proximal angle of the fibular rotation was 0.2° and distal 0.4°. The interindividual variations of the angles and distances exceeded the intraindividual values partly by 3 to 4 fold. CONCLUSIONS: Within the scope of this study neither the tibiofibular distance, nor the tibiofibular angle changed significantly through the different joint positions. The intraindividual differences were little while the interindividual variations of the parameters were distinctive.

Early alterations in hippocampal perisomatic GABAergic synapses and network oscillations in a mouse model of Alzheimer's disease amyloidosis.

Several lines of evidence imply changes in inhibitory interneuron connectivity and subsequent alterations in oscillatory network activities in the pathogenesis of Alzheimer's Disease (AD). Recently, we provided evidence for an increased immunoreactivity of both the postsynaptic scaffold protein gephyrin and the GABAA receptor γ2-subunit in the hippocampus of young (1 and 3 months of age), APPPS1 mice. These mice represent a well-established model of cerebral amyloidosis, which is a hallmark of human AD. In this study, we demonstrate a robust increase of parvalbumin immunoreactivity and accentuated projections of parvalbumin positive (PV+) interneurons, which target perisomatic regions of pyramidal cells within the hippocampal subregions CA1 and CA3 of 3-month-old APPPS1 mice. Colocalisation studies confirmed a significant increase in the density of PV+ projections labeled with antibodies against a presynaptic (vesicular GABA transporter) and a postsynaptic marker (gephyrin) of inhibitory synapses within the pyramidal cell layer of CA1 and CA3. As perisomatic inhibition by PV+-interneurons is crucial for the generation of hippocampal network oscillations involved in spatial processing, learning and memory formation we investigated the impact of the putative enhanced perisomatic inhibition on two types of fast neuronal network oscillations in acute hippocampal slices: 1. spontaneously occurring sharp wave-ripple complexes (SPW-R), and 2. cholinergic γ-oscillations. Interestingly, both network patterns were generally preserved in APPPS1 mice similar to WT mice. However, the comparison of simultaneous CA3 and CA1 recordings revealed that the incidence and amplitude of SPW-Rs were significantly lower in CA1 vs CA3 in APPPS1 slices, whereas the power of γ-oscillations was significantly higher in CA3 vs CA1 in WT-slices indicating an impaired communication between the CA3 and CA1 network activities in APPPS1 mice. Taken together, our data demonstrate an increased GABAergic synaptic output of PV+ interneurons impinging on pyramidal cells of CA1 and CA3, which might limit the coordinated cross-talk between these two hippocampal areas in young APPPS1 mice and mediate long-term changes in synaptic inhibition during progression of amyloidosis.

Targeted Depletion of Primary Cilia in Dopaminoceptive Neurons in a Preclinical Mouse Model of Huntington's Disease.

Multiple pathomechanisms triggered by mutant Huntingtin (mHTT) underlie progressive degeneration of dopaminoceptive striatal neurons in Huntington's disease (HD). The primary cilium is a membrane compartment that functions as a hub for various pathways that are dysregulated in HD, for example, dopamine (DA) receptor transmission and the mechanistic target of rapamycin (mTOR) pathway. The roles of primary cilia (PC) for the maintenance of striatal neurons and in HD progression remain unknown. Here, we investigated PC defects in vulnerable striatal neurons in a progressive model of HD, the mHTT-expressing knock-in zQ175 mice. We found that PC length is affected in striatal but not in cortical neurons, in association with the accumulation of mHTT. To explore the role of PC, we generated conditional mutant mice lacking IFT88, a component of the anterograde intraflagellar transport-B complex lacking PC in dopaminoceptive neurons. This mutation preserved the expression of the dopamine 1 receptor (D1R), and the survival of striatal neurons, but resulted in a mild increase of DA metabolites in the striatum, suggesting an imbalance of ciliary DA receptor transmission. Conditional loss of PC in zQ175 mice did not trigger astrogliosis, however, mTOR signaling was more active and resulted in a more pronounced accumulation of nuclear inclusions containing mHTT. Further studies will be required of aged mice to determine the role of aberrant ciliary function in more advanced stages of HD.

Gephyrin: a key regulatory protein of inhibitory synapses and beyond.

Scaffolding proteins underlying postsynaptic membrane specializations are important structural and functional components of both excitatory and inhibitory synapses. At inhibitory synapses, gephyrin was identified as anchoring protein. Gephyrin self-assembles into a complex flat submembranous lattice that slows the lateral mobility of glycine and GABAA receptors, thus allowing for their clustering at postsynaptic sites. The structure and stability of the gephyrin lattice is dynamically regulated by posttranslational modifications and interactions with binding partners. As gephyrin is the core scaffolding protein for virtually all inhibitory synapses, any changes in the structure or stability of its lattice can profoundly change the packing density of inhibitory receptors and, therefore, alter inhibitory drive. Intriguingly, gephyrin plays a completely independent role in non-neuronal cells, where it facilitates two steps in the biosynthesis of the molybdenum cofactor. In this review, we provide an overview of the role of gephyrin at inhibitory synapses and beyond. We discuss its dynamic regulation, the nanoscale architecture of its synaptic lattice, and the implications of gephyrin dysfunction for neuropathologic conditions, such as Alzheimer's disease and epilepsy.

Causal Interrogation of Neuronal Networks and Behavior through Virally Transduced Ivermectin Receptors.

The causal interrogation of neuronal networks involved in specific behaviors requires the spatially and temporally controlled modulation of neuronal activity. For long-term manipulation of neuronal activity, chemogenetic tools provide a reasonable alternative to short-term optogenetic approaches. Here we show that virus mediated gene transfer of the ivermectin (IVM) activated glycine receptor mutant GlyRα1 (AG) can be used for the selective and reversible silencing of specific neuronal networks in mice. In the striatum, dorsal hippocampus, and olfactory bulb, GlyRα1 (AG) promoted IVM dependent effects in representative behavioral assays. Moreover, GlyRα1 (AG) mediated silencing had a strong and reversible impact on neuronal ensemble activity and c-Fos activation in the olfactory bulb. Together our results demonstrate that long-term, reversible and re-inducible neuronal silencing via GlyRα1 (AG) is a promising tool for the interrogation of network mechanisms underlying the control of behavior and memory formation.

Biphasic Alteration of the Inhibitory Synapse Scaffold Protein Gephyrin in Early and Late Stages of an Alzheimer Disease Model.

The pathogenesis of Alzheimer disease (AD) is thought to begin many years before the diagnosis of dementia. Accumulating evidence indicates the involvement of GABAergic neurotransmission in the physiopathology of AD. However, in comparison to excitatory synapses, the structural and functional alterations of inhibitory synapses in AD are less well characterized. We studied the expression and distribution of proteins specific for inhibitory synapses in hippocampal areas of APPPS1 mice at different ages. Interestingly, by immunoblotting and confocal fluorescence microscopy, we disclosed a robust increase in the expression of gephyrin, an organizer of ligand-gated ion channels at inhibitory synapses in hippocampus CA1 and dentate gyrus of young presymptomatic APPPS1 mice (1 to 3 months) as compared to controls. The postsynaptic γ2-GABA(A)-receptor subunit and the presynaptic vesicular inhibitory amino acid transporter protein showed similar expression patterns. In contrast, adult transgenic animals (12 months) displayed decreased levels of these proteins in comparison to wild type in hippocampus areas devoid of amyloid plaques. Within most plaques, strong gephyrin immunoreactivity was detected, partially colocalizing with vesicular amino acid transporter and GABA(A)-receptor γ2 subunit immunoreactivities. Our results indicate a biphasic alteration in expression of hippocampal inhibitory synapse components in AD. Altered inhibition of neurotransmission might be an early prognostic marker and might even be involved in the pathogenesis of AD.

Forebrain-specific loss of synaptic GABAA receptors results in altered neuronal excitability and synaptic plasticity in mice.

Mutations that result in the defective trafficking of γ2 subunit containing GABAA receptors (γ2-GABAARs) are known to reduce synaptic inhibition. Whether perturbed clustering of non-mutated GABAARs similarly reduces synaptic inhibition in vivo is less clear. In this study we provide evidence that the loss of postsynaptic γ2-GABAARs upon postnatal ablation of gephyrin, the major scaffolding protein of inhibitory postsynapses, from mature principal neurons within the forebrain results in reduced induction of long-term potentiation (LTP) and impaired network excitability within the hippocampal dentate gyrus. The preferential reduction in not only synaptic γ2-GABAAR cluster number at dendritic sites but also the decrease in γ2-GABAAR density within individual clusters at dendritic inhibitory synapses suggests that distal synapses are more sensitive to the loss of gephyrin expression than proximal synapses. The fact that these mice display behavioural features of anxiety and epilepsy emphasises the importance of postsynaptic γ2-GABAAR clustering for synaptic inhibition.