Synthesis and discovery of the putative cognitive enhancer BRS-015: effect on glutamatergic transmission and synaptic plasticity

This thesis is concerned with the discovery of a novel heterocyclic compound – BRS-015, its synthesis and an analysis of its effects on excitatory synaptic transmission at a major pathway in the brain. BRS-015 is related to the natural product clausenamide, which has been shown to facilitate synaptic transmission. As such, clausenamide and related analogues may possess therapeutic potential as memory enhancing drugs, which are in urgent need of development due to the increasing numbers of patients diagnosed with memory disorders and for which there is no current effective therapy. BRS-015 was synthesized using a novel approach to the core structure of clausenamide involving an intramolecular acylal cyclisation reaction, which has not previously been reported. The first section of the thesis opens with a description of the discovery, structure and biological activity of clausenamide and discussion of previous synthetic strategies adopted by a number of research groups and attempts to classify these into the varying approaches towards the central core of clausenamide. The second section describes the structure of the rat brain and the types of processes involved in memory formation, as well as the neurophysiological assays used to investigate synaptic transmission and plasticity. The second group of chapters describes our own approach to the core of clausenamide and the synthesis of BRS-015, with a detailed discussion of the structural analysis and investigation of the intramolecular acylal cyclisation reaction used during the synthetic process. The third chapter describes the neurophysiological assays used in our investigations into the effects of BRS-015, which was tested against glutamatergic synaptic transmission and plasticity in acute rat hippocampal slices. BRS-015 was shown to reversibly enhance the amplitude of AMPA receptor mediated EPSCs recorded from CA3 pyramidal neurones and evoked by dentate stimulation. When tested in the presence of selective glutamate receptor antagonists, BRS-015 did not have this powerful enhancing effect on kainate or NMDA receptor mediated EPSCs. In addition, BRS-015 increased the amplitude of glutamate-evoked currents in CA3 pyramidal neurones and did not alter short-term synaptic plasticity but facilitated the induction of mossy fibre LTP, with little effect at associational/commissural synapses. BRS-015 has striking enhancing properties on AMPA receptor mediated synaptic transmission at mossy fibre synapses either by directly interacting with AMPA receptors or via indirect modulation, the mechanisms of which could lead to synapse strengthening

SNX27-Mediated Recycling of Neuroligin-2 Regulates Inhibitory Signaling

GABAA receptors mediate fast inhibitory transmission in the brain, and their number can be rapidly up- or downregulated to alter synaptic strength. Neuroligin-2 plays a critical role in the stabilization of synaptic GABAA receptors and the development and maintenance of inhibitory synapses. To date, little is known about how the amount of neuroligin-2 at the synapse is regulated and whether neuroligin-2 trafficking affects inhibitory signaling. Here, we show that neuroligin-2, when internalized to endosomes, co-localizes with SNX27, a brain-enriched cargo-adaptor protein that facilitates membrane protein recycling. Direct interaction between the PDZ domain of SNX27 and PDZ-binding motif in neuroligin-2 enables membrane retrieval of neuroligin-2, thus enhancing synaptic neuroligin-2 clusters. Furthermore, SNX27 knockdown has the opposite effect. SNX27-mediated up- and downregulation of neuroligin-2 surface levels affects inhibitory synapse composition and signaling strength. Taken together, we show a role for SNX27-mediated recycling of neuroligin-2 in maintenance and signaling of the GABAergic synapse

A Common Precursor Approach to Structurally Diverse Natural Products: The Synthesis of the Core Structure of (±)-Clausenamide and the Total Synthesis of (±)-Hyalodendrin

Structurally diverse natural products from unrelated sources typically require the development of individual synthetic routes. In a novel approach, we have shown that the epidithiodiketopiperazine-derived natural product (±)-hyalodendrin and the core structure of the unrelated pyrrolidine-derived natural product clausenamide can be synthesised from a common synthetic precursor in good yield by simple variation of the reaction conditions

Physiological signature of a novel potentiator of AMPA receptor signalling.

We have synthesized a novel small molecule based on the pyrrolidinone-containing core structure of clausenamide, which is a candidate anti-dementia drug. The synthetic route yielded multi-gram quantities of an isomeric racemate mixture in a short number of steps. When tested in hippocampal slices from young adult rats the compound enhanced AMPA receptor-mediated signalling at mossy fibre synapses, and potentiated inward currents evoked by local application of l-glutamate onto CA3 pyramidal neurons. It facilitated the induction of mossy fibre LTP, but the magnitude of potentiation was smaller than that observed in untreated slices. The racemic mixture was separated and it was shown that only the (-) enantiomer was active. Toxicity analysis indicated that cell lines tolerated the compound at concentrations well above those enhancing synaptic transmission. Our results unveil a small molecule whose physiological signature resembles that of a potent nootropic drug

A Short Total Synthesis of (±)-Lycorane by a Sequential Intramolecular Acylal Cyclisation (IAC) and Intramolecular Heck Addition Reaction

An intramolecular acylal cyclisation (IAC) approach to the synthesis of a range of bicyclic heterocycles is reported. As an example of the utility of the IAC reaction, the methodology was applied in a protecting-group free five-step total synthesis of (±)-lycorane, incorporating a novel intramolecular Heck addition reaction to generate the pentacyclic core structure of the natural product in good yield

Three-Dimensional Printing of a Scalable Molecular Model and Orbital Kit for Organic Chemistry Teaching and Learning

Three-dimensional (3D) chemical models are a well-established learning tool used to enhance the understanding of chemical structures by converting two-dimensional paper or screen outputs into realistic three-dimensional objects. While commercial atom model kits are readily available, there is a surprising lack of large molecular and orbital models that could be used in large spaces. As part of a program investigating the utility of 3D printing in teaching, a modular size-adjustable molecular model and orbital kit was developed and produced using 3D printing and was used to enhance the teaching of stereochemistry, isomerism, hybridization, and orbitals

STUDI DESKRIPTIF LEVEL BERPIKIR GEOMETRI VAN HIELE SISWA DI SMP NEGERI PERCONTOHAN DI LEMBANG

Geometri sekolah mempunyai peluang besar untuk dipahami oleh siswa dibandingkan dengan cabang ilmu matematika yang lainnya. Hal ini dikarenakan pengenalan konsep dasar geometri sudah dikenal oleh siswa sejak usia dini, seperti mengenal bangun-bangun geometri. Namun beberapa penelitian menunjukkan bahwa masih banyak siswa yang mengalami kesulitan dalam belajar geeometri, khususnya pada tingkat SMP. Oleh karena itu diperlukan penelitian terhadap level berpikir geometri siswa. Penelitian ini bertujuan untuk mengetahui: (1) level berpikir geometri siswa di SMP Negeri percontohan di Lembang, dan (2) menelaah apakah pembelajaran geometri yang berlangsung di sekolah menerapkan tahapan pembelajaran Van Hiele atau tidak. Metode dalam penelitian ini merupakan studi deskriptif dengan subjek penelitian adalah siswa kelas IX dari dua sekolah menengah pertama di Lembang. Instrumen dalam penelitian ini terdiri dari: (1) instrumen tes, yaitu tes level berpikir geometri Van Hiele pada materi bangun datar. Hasil dari tes ini dianalisis dengan kategori level berpikir sebagai berikut: level 0 adalah tahap pengenalan; level 1 adalah tahap analisis; level 2 adalah tahap pengurutan; level 3 adalah tahap deduksi formal; dan level 4 adalah tahap akurasi. (2) Instrumen non tes, yaitu berupa wawancara terhadap guru dan murid. Berdasarkan hasil penelitian diperoleh kesimpulan bahwa: (1) secara keseluruhan siswa SMP telah memasuki tahap berpikir geometri Van Hiele. Sebagian besar siswa berada pada tahap pengenalan (level 0) yaitu 81,16%, sedangkan sisanya telah memasuki tahap analisis (level 1) sebesar 17,39% dan tahap pengurutan (level 2) sebesar 1,45%. (2) Pembelajaran geometri di sekolah kurang memperhatikan tahapan pembelajaran geometri Van Hiele---------- Student has a big opportunity to understand geometry because the basic concept has early familiar, such as know the geometry’s objects. However, some of the research were show that many student difficult to learn geometry, specifically for junior high school. Because of that, it necessary to research about the geometry level thinking. The goal of the research are to know: (1) student geometry level thinking at the model of junior high school in Lembang, and (2) observe the lesson geometry at school by use the phase of Van Hiele geometry learning. The method is descriptive study with the subject are the student from IX class of two junior high school in Lembang. The instrument is: (1) test instrument, is Van Hiele geometry level test. The result will be analysis by categories of Van Hiele: level 0 is visualization; level 1 is analysis; level 2 is informal deduction; level 3 is deduction; and level 4 is rigor. (2) Non-test instrument, is interview to the teacher and student. Base of the research, the conclusion are: (1) by and large the student has include the Van Hiele geometry level. Student at level 0 is 81, 16%, at level 1 is 17,3% and at level 2 is 1,45%. (2) School did’nt use the phase of Van Hiele geometry learning

Serum levels of osteoprotegerin and receptor activator of nuclear factor -κB ligand in children with early juvenile idiopathic arthritis: a 2-year prospective controlled study

<p>Abstract</p> <p>Background</p> <p>The clinical relevance of observations of serum levels of osteoprotegerin (OPG) and receptor activator of nuclear factor -κB ligand (RANKL) in juvenile idiopathic arthritis (JIA) is not clear. To elucidate the potential role of OPG and RANKL in JIA we determined serum levels of OPG and RANKL in patients with early JIA compared to healthy children, and prospectively explored changes in relation to radiographic score, bone and lean mass, severity of the disease, and treatment.</p> <p>Methods</p> <p>Ninety children with early oligoarticular or polyarticular JIA (ages 6-18 years; mean disease duration 19.4 months) and 90 healthy children individually matched for age, sex, race, and county of residence, were examined at baseline and 2-year follow-up. OPG and RANKL were quantified by enzyme-immunoassay. Data were analyzed with the use of t-tests, ANOVA, and multiple regression analyses.</p> <p>Results</p> <p>Serum OPG was significantly lower in patients than controls at baseline, and there was a trend towards higher RANKL and a lower OPG/RANKL ratio. Patients with polyarthritis had significantly higher increments in RANKL from baseline to follow-up, compared to patients with oligoarthritis. RANKL was a significant negative predictor for increments in total body lean mass. Patients who were receiving corticosteroids (CS) or disease-modifying antirheumatic drugs (DMARDs) at follow-up had higher OPG/RANKL ratio compared with patients who did not receive this medication.</p> <p>Conclusions</p> <p>The data supports that levels of OPG are lower in patients with JIA compared to healthy children, and higher levels of RANKL is associated with more serious disease. RANKL was a significant negative predictor of lean mass in patients with JIA. The OPG/RANKL ratio was higher in patients on DMARDs or CS treatment.</p

Miro1-dependent mitochondrial dynamics in parvalbumin interneurons

Parvalbumin (PV+) interneurons constitute a small proportion of the total neuronal population (less than 2% in the hippocampus), yet they possess crucial roles in shaping neuronal network activity (Freund and Buzsáki, 1996; Jonas et al., 2004; Pelkey et al., 2017). PV+ interneurons inhibit their postsynaptic targets efficiently by applying fast perisomatic inhibition and have been directly implicated in the generation of network activity at the gamma (γ) band frequency (30–80 Hz) (Antonoudiou et al., 2020; Cardin et al., 2009; Hájos et al., 2004; Mann et al., 2005; Sohal et al., 2009). Network oscillations at γ-band frequency are believed to facilitate information transmission through circuit synchronization and local gain control that may be instrumental in multiple cognitive processes such as attention, learning, and memory (Akam and Kullmann, 2010; Fries, 2015; Howard et al., 2003; Montgomery and Buzsaki, 2007; Sohal, 2016). Importantly, these oscillations are thought to be metabolically very costly, and it has therefore been postulated that PV+ interneurons require substantial amounts of energy via ATP hydrolysis to sustain the high firing rate and dissipate ion gradients during neuronal transmission (Attwell and Laughlin, 2001; Kann, 2011; Kann, 2016; Kann and Kovács, 2007; Kann et al., 2014). Thus, it is crucial to understand the metabolic expenditure and the involvement of mitochondria in PV+ interneurons. Indeed, electron microscopy, histochemical, and transcriptomic approaches have revealed that PV+ interneurons have a higher density of energy-producing mitochondria and elevated expression levels of electron transport chain components (Adams et al., 2015; Gulyás et al., 2006; Nie and Wong-Riley, 1995; Paul et al., 2017). The spatiotemporal organization of mitochondria is essential for the precise provision of ATP and Ca2+-buffering for neuronal transmission and communication (Devine and Kittler, 2018; MacAskill and Kittler, 2010). Miro1 is a mitochondrial adaptor protein, responsible for coupling mitochondria to the cytoskeleton and for their bidirectional trafficking in axons and dendrites (Birsa et al., 2013; Guo et al., 2005; López-Doménech et al., 2016; López-Doménech et al., 2018; Macaskill et al., 2009; Nguyen et al., 2014; Saotome et al., 2008; Wang and Schwarz, 2009). Global deletion of Miro1 (encoded by the Rhot1 gene) is perinatal lethal, while the conditional removal of Miro1 from cortical and hippocampal pyramidal cells alters the occupancy of dendritic mitochondria due to impairment in trafficking, resulting in dendritic degeneration and cell death (López-Doménech et al., 2016). In contrast, the significance of mitochondrial trafficking and distribution in PV+ interneurons, and the role of Miro1, is completely unexplored and especially interesting as their axon is highly branched with a cumulative length reaching up to 50 mm in the hippocampus (Hu et al., 2014). In this study, we generated a transgenic mouse line where mitochondria are fluorescently labelled in PV+ interneurons. We crossed this line with the Miro1 floxed mouse (Rhot1flox/flox), to generate a model where Miro1 was conditionally knocked-out exclusively in PV+ interneurons. Using two-photon live-imaging of ex vivo organotypic brain slices, we demonstrated a reduction in mitochondrial trafficking in the absence of Miro1 in PV+ interneurons in the hippocampus. The impairment in Miro1-directed mitochondrial transport led to an accumulation of mitochondria in the soma and their depletion from axonal presynaptic terminals in acute hippocampal brain slices. Loss of Miro1 resulted in alterations in axonal but not dendritic branching in PV+ interneurons. While the ability of PV+ interneurons to apply long-lasting inhibition to post-synaptic targets remained intact, the changes in Miro1-dependent mitochondrial dynamics were accompanied by an increased frequency of γ-oscillations in hippocampal brain slices and a reduction in anxiety-related emotional behavior. Thus, we show that Miro1-dependent mitochondrial positioning is essential for correct PV+ interneuron function, network activity, and anxiolytic animal behavior