Identification of New Drug Candidates Against \u3cem\u3eBorrelia burgdorferi\u3c/em\u3e Using High-Throughput Screening

Lyme disease is the most common zoonotic bacterial disease in North America. It is estimated that .300,000 cases per annum are reported in USA alone. A total of 10%–20% of patients who have been treated with antibiotic therapy report the recrudescence of symptoms, such as muscle and joint pain, psychosocial and cognitive difficulties, and generalized fatigue. This condition is referred to as posttreatment Lyme disease syndrome. While there is no evidence for the presence of viable infectious organisms in individuals with posttreatment Lyme disease syndrome, some researchers found surviving Borrelia burgdorferi population in rodents and primates even after antibiotic treatment. Although such observations need more ratification, there is unmet need for developing the therapeutic agents that focus on removing the persisting bacterial form of B. burgdorferi in rodent and nonhuman primates. For this purpose, high-throughput screening was done using BacTiter-Glo assay for four compound libraries to identify candidates that stop the growth of B. burgdorferi in vitro. The four chemical libraries containing 4,366 compounds (80% Food and Drug Administration [FDA] approved) that were screened are Library of Pharmacologically Active Compounds (LOPAC1280), the National Institutes of Health Clinical Collection, the Microsource Spectrum, and the Biomol FDA. We subsequently identified 150 unique compounds, which inhibited .90% of B. burgdorferi growth at a concentration of ,25 µM. These 150 unique compounds comprise many safe antibiotics, chemical compounds, and also small molecules from plant sources. Of the 150 unique compounds, 101 compounds are FDA approved. We selected the top 20 FDA-approved molecules based on safety and potency and studied their minimum inhibitory concentration and minimum bactericidal concentration. The promising safe FDA-approved candidates that show low minimum inhibitory concentration and minimum bactericidal concentration values can be chosen as lead molecules for further advanced studies

Screening of NCI-DTP Library to Identify New Drug Candidates for \u3cem\u3eBorrelia burgdorferi\u3c/em\u3e

Lyme disease is the most rapidly growing tick borne zoonotic disease of the Northern Hemisphere and is among the 10 most commonly reported nationally notifiable diseases in the United States.1 Clinical presentations include erythema migrans, fever, chills, muscle and joint pain.2, 3 Though these symptoms tend to fade away even without therapeutic intervention, a significant number of untreated patients develop arthritis and persistent myalgia following exposure to Borrelia burgdorferi.4 Furthermore, 10–20% of patients treated for Lyme disease develop symptoms considered typical, or even exaggerated, including muscle, joint pain and generalized fatigue5, 6. This condition is referred as post-treatment lyme disease syndrome (PTLDS)

"Bruchpilot" - Molekulare und funktionelle Charakterisierung eines neuen Proteins der aktiven Zone der Drosophila-Synapse

Chemical neurotransmission is a complex process of central importance for nervous system function. It is thought to be mediated by the orchestration of hundreds of proteins for its successful execution. Several synaptic proteins have been shown to be relevant for neurotransmission and many of them are highly conserved during evolution- suggesting a universal mechanism for neurotransmission. This process has checkpoints at various places like, neurotransmitter uptake into the vesicles, relocation of the vesicles to the vicinity of calcium channels in order to facilitate Ca2+ induced release thereby modulating the fusion probability, formation of a fusion pore to release the neurotransmitter and finally reuptake of the vesicles by endocytosis. Each of these checkpoints has now become a special area of study and maintains its own importance for the understanding of the overall process. Ca2+ induced release occurs at specialized membrane structures at the synapse known as the active zones. These are highly ordered electron dense grids and are composed of several proteins which assist the synaptic vesicles in relocating in the vicinity of Ca2+ channels thereby increasing their fusion probability and then bringing about the vesicular fusion itself. All the protein modules needed for these processes are thought to be held in tight arrays at the active zones, and the functions of a few have been characterized so far at the vertebrate active zones. Our group is primarily interested in characterizing the molecular architecture of the Drosophila synapse. Due to its powerful genetics and well-established behavioural assays Drosophila is an excellent system to investigate neuronal functioning. Monoclonal antibodies (MABs) from a hybridoma library against Drosophila brain are routinely used to detect novel proteins in the brain in a reverse genetic approach. Upon identification of the protein its encoding genetic locus is characterized and a detailed investigation of its function is initiated. This approach has been particularly useful to detect synaptic proteins, which may go undetected in a forward genetic approach due to lack of an observable phenotype. Proteins like CSP, Synapsin and Sap47 have been identified and characterized using this approach so far. MAB nc82 has been one of the shortlisted antibodies from the same library and is widely used as a general neuropil marker due to the relative transparency of immunohistochemical whole mount staining obtained with this antibody. A careful observation of double stainings at the larval neuromuscular junctions with MAB nc82 and other pre and post-synaptic markers strongly suggested an active zone localization of the nc82 antigen. Synaptic architecture is well characterized in Drosophila at the ultrastructural level. However, molecular details for many synaptic components and especially for the active zone are almost entirely unknown. A possible localization at the active zone for the nc82 antigen served as the motivation to initiate its biochemical characterization and the identification of the encoding gene. In the present thesis it is shown by 2-D gel analysis and mass spectrometry that the nc82 antigen is a novel active zone protein encoded by a complex genetic locus on chromosome 2R. By RT-PCR exons from three open reading frames previously annotated as separate genes are demonstrated to give rise to a transcript of at least 5.5 kb. Northern blots produce a prominent signal of 11 kb and a weak signal of 2 kb. The protein encoded by the 5.5 kb transcript is highly conserved amongst insects and has at its N-terminus significant homology to the previously described vertebrate active zone protein ELKS/ERC/CAST. Bioinformatic analysis predicts coiled-coil domains spread all over the sequence and strongly suggest a function involved in organizing or maintaining the structure of the active zone. The large C-terminal region is highly conserved amongst the insects but has no clear homologues in veretebrates. For a functional analysis of this protein transgenic flies expressing RNAi constructs under the control of the Gal4 regulated enhancer UAS were kindly provided by the collaborating group of S.Sigrist (Gِttingen). A strong pan-neuronal knockdown of the nc82 antigen by transgenic RNAi expression leads to embryonic lethality. A relatively weaker RNAi expression results in behavioural deficits in adult flies including unstable flight and impaired walking behavior. Due to this peculiar phenotype as observed in the first knockdown studies the gene was named “bruchpilot” (brp) encoding the protein “Bruchpilot (BRP)” (German for crash pilot). A pan-neuronal as well as retina specific downregulation of this protein results in loss of ON and OFF transients in ERG recordings indicating dysfunctional synapses. Retina specific downregulation also shows severely impaired optomotor behaviour. Finally, at an ultrastructural level BRP downregulation seems to impair the formation of the characteristic T-shaped synaptic ribbons at the active zones without significantly altering the overall synaptic architecture (in collaboration with E.Asan). Vertebrate active zone protein Bassoon is known to be involved in attaching the synaptic ribbons to the active zones as an adapter between active zone proteins RIBEYE and ERC/CAST. A mutation in Bassoon results in a floating synaptic ribbon phenotype. No protein homologous to Bassoon has been observed in Drosophila. BRP downregulation also results in absence of attached synaptic ribbons at the active zones. This invites the speculation of an adapter like function for BRP in Drosophila. However, while Bassoon mutant mice are viable, BRP deficit in addition to the structural phenotype also results in severe behavioural and physiological anomalies and even stronger downregulation causes embryonic lethality. This therefore suggests an additional and even more important role for BRP in development and normal functioning of synapses in Drosophila and also in other insects. However, how BRP regulates synaptic transmission and which other proteins are involved in this BRP dependant pathway remains to be investigated. Such studies certainly will attract prominent attention in the future.Die chemische Signalübertragung an Synapsen ist ein komplexer Prozess mit zentraler Bedeutung für die Funktion von Nervensystemen. Man nimmt an, dass er auf einem Zusammenspiel hunderter verschiedener Proteine beruht. Diverse Synopsenproteine haben sich für die Neurotransmission als relevant erwiesen und viele davon sind in der Evolution hoch konserviert, was einen universalen Mechanismus der Neurotransmission wahrscheinlich macht. Dieser Prozess ist in zahlreiche aufeinander folgende Schritte unterteilt, wie die Neurotransmitteraufnahme in Vesikel, den Transport von Vesikeln in die Nنhe von Calciumkanنlen, die Ausbildung einer Fusionspore zur Transmitterausschüttung und schlieكlich die Wiederaufnahme von Vesikeln durch Endozytose. Jeder dieser Teilschritte wird momentan gezielt erforscht und spielt für sich genommen eine zentrale Rolle für das Verstنndnis des gesamten Prozesses. Die Calcium-induzierte Transmitterausschüttung findet an spezialisierten Membranstrukturen der Synapsen statt, den aktiven Zonen. Diese sind hoch organisierte, elektronendichte Gitterstrukturen und bestehen aus verschiedenen Proteinen, die den synaptischen Vesikeln bei der Verlagerung in die Nنhe von Calciumkanنlen behilflich sind. Alle Proteinmodule, die für diese Prozesse nِtig sind, scheinen eng aneinandergereiht an den aktiven Zonen vorzuliegen. Nur von wenigen konnte bisher bei Vertebraten die Funktion an der aktiven Zone charakterisiert werden. Ein Fokus der Arbeitsgruppe, an der diese Doktorarbeit durchgeführt wurde, besteht in der Charakterisierung des molekularen Aufbaus der Synapse von Drosophila. Die Taufliege ist aufgrund eines reichen Angebots hِchsteffektiver genetischer Methoden und vielfنltiger Verhaltensparadigmen ein exzellentes Modellsystem, um die neuronale Signalübertragung zu untersuchen. Monoklonale Antikِrper (MAKs) aus einer Hybridomabank gegen das Drosophila Gehirn werden standardmنكig verwendet, um neue Gehirnproteine mittels der „reverse genetics“- Methode zu identifizieren. Dazu wird der entsprechende genetische Lokus charakterisiert und eine detaillierte Untersuchung der Proteinfunktion initiiert. Diese Vorgehensweise war besonders hilfreich bei der Identifizierung von Synapsenproteinen, die bei der „forward genetics“-Methode aufgrund des Fehlens eines beobachtbaren Phنnotyps übersehen würden. Proteine wie CSP, Synapsin und Sap47 wurden so gefunden und charakterisiert. I MAK nc82 stammt aus dieser Hybridomabank und wird in vielen Labors als allgemeiner Neuropilmarker aufgrund seiner hervorragenden Fنrbungseigenschaften in Gehirnprنparaten verwendet. Doppelfنrbungen der larvalen neuromuskulنren Synapse mit dem Antikِrper nc82 in Kombination mit anderen prن- und postsynaptischen Markern deuteten stark auf eine Lokalisierung des Antigens an der aktiven Zone hin. Die Synapsenarchitektur von Drosophila ist auf der ultrastrukturellen Ebene gut verstanden. Jedoch sind die molekularen Details vieler Synapsenkomponenten, besonders die der aktiven Zone, nicht bekannt. Die vermutete Lokalisierung des nc82 Antigens an der aktiven Zone war daher der Ansatzpunkt, eine biochemische Charakterisierung zu initiieren und das entsprechende Gen zu identifizieren. In der vorliegenden Arbeit wird durch 2-D Gelelektrophorese und Massenspektrometrie gezeigt, das das nc82 Antigen ein neues Protein der aktiven Zone ist, welches von einem komplexen Genlokus auf Chromosom 2R kodiert wird. Durch RT-PCR wurde gezeigt, dass die Exons von drei offenen Leserastern, die bisher als getrennte Gene annotiert wurden, ein Transkript von mindestens 5,5 kb Lنnge kodieren. Northern Blots ergaben ein deutliches Signal bei 11 kb und ein schwنcheres bei 2 kb. Das von dem 5,5 kb Transkript resultierende Protein ist hoch konserviert in der Gruppe der Insekten und weist an seiner N-terminalen Domنne eine signifikante Homologie zu den bisher beschriebenen Vertebratenproteinen der aktiven Zone ELKS/ERC/CAST auf. Bioinformatische Analysen sagen „coiled-coil“ Domنnen vorher, die über die gesamte Sequenz verteilt sind. Dies deutet stark auf eine Funktion bei der Organisation oder der Aufrechterhaltung der prنsynaptischen Struktur hin. Die groكe C-terminale Region ist zwar bei Insekten hoch konserviert, zeigt aber keine eindeutige Homologie zu Proteinen von Vertebraten. Für die Funktionsanalyse dieses Proteins wurden transgene Fliegen, die UAS-RNAi Konstrukte in ihrem Genom tragen und durch entsprechende GAL4-Linien getrieben werden kِnnen, freundlicherweise von der kollaborierenden Arbeitsgruppe von S. Sigrist (Gِttingen) zur Verfügung gestellt. Der pan-neuronale „knock-down“ des nc82 Antigens durch transgene RNAi-Expression führt zu embryonaler Letalitنt. Eine schwنchere RNAi-Expression führt bei adulten Fliegen zu Verhaltensdefekten, wie instabilem Flug und beeintrنchtigtem Laufverhalten. Aufgrund dieser Phنnotypen, die in den ersten „knock-down“ Studien beobachtet wurden, wurde das Gen „bruchpilot“ (brp) und das zugehِrige Protein „Bruchpilot“ (BRP) genannt. Die pan-neuronale, sowie die retinaspezifische Reduktion des Proteins führt zu einem Verlust der ON und OFF Transienten des Elektroretinogramms, was auf nichtfunktionelle Synapsen hindeutet. Die retinaspezifische Reduktion des Proteins hat eine Beeintrنchtigung der optomotorischen Reaktion zur Folge. Auكerdem scheint auf der ultrastrukturellen Ebene die Bildung der charakteristischen T-fِrmigen „ribbons“ der aktiven Zonen beeintrنchtigt zu sein, jedoch ohne signifikante Verنnderungen der Gesamtarchitektur der Synapse (in Kollaboration mit E. Asan). Von Basson, einem Protein der aktiven Zone bei Vertebraten, ist bekannt, dass es an der Anheftung der synaptischen „ribbons“ an den aktiven Zonen beteiligt ist. Es fungiert als Adapter zwischen RIBEYE und ELKS/ERC/CAST, zwei weiteren Proteinen der aktiven Zone. Die Mutation von Bassoon hat zur Folge, dass die synaptischen „ribbons“ frei im Zytoplasma treiben. Für Bassoon ist kein homologes Drosophila-Protein bekannt. Die Reduktion von BRP bedingt ebenfalls ein Fehlen befestigter „ribbons“ an der aktiven Zone. Dies kِnnte auf eine Art Adapterfunktion von BRP hindeuten. Jedoch hat das Fehlen von BRP zusنtzlich zum strukturellen Phنnotyp auch deutliche Verhaltensabnormalitنten und starke physiologische Beeintrنchtigungen zur Folge. Eine noch stنrkere Reduktion bedingt auكerdem embryonale Lethalitنt, wohingegen Mausmutanten ohne Bassoon lebensfنhig sind. Daraus ergibt sich, dass BRP eine weitere, wichtige Rolle wنhrend der Entwicklung und für die Funktion von Synapsen bei Drosophila und mِglicherweise auch bei anderen Insekten einnimmt. Es muss aber noch geklنrt werden, auf welche Weise BRP die synaptische Signalübertragung reguliert und welche anderen Proteine in diesem BRP-abhنngigen Pfad involviert sind. Derartige Studien werden mit Sicherheit in der Zukunft eine bedeutende Rolle spielen

A critical role for the PAR-1/MARK-tau axis in mediating the toxic effects of Aβ on synapses and dendritic spines

Alzheimer's disease (AD) is the most common neurodegenerative disease and the leading cause of dementia in the elderly. Accumulating evidence supports soluble amyloid-β (Aβ) oligomers as the leading candidate for the causative agent in AD and synapses as the primary site of Aβ oligomer action. However, the molecular and cellular mechanisms by which Aβ oligomers cause synaptic dysfunction and cognitive impairments remain poorly understood. Using primary cultures of rat hippocampal neurons as a model system, we show that the partitioning defective-1 (PAR-1)/microtubule affinity-regulating kinase (MARK) family kinases act as critical mediators of Aβ toxicity on synapses and dendritic spines. Overexpression of MARK4 led to tau hyperphosphorylation, reduced expression of synaptic markers, and loss of dendritic spines and synapses, phenotypes also observed after Aβ treatment. Importantly, expression of a non-phosphorylatable form of tau with the PAR-1/MARK site mutated blocked the synaptic toxicity induced by MARK4 overexpression or Aβ treatment. To probe the involvement of endogenous MARK kinases in mediating the synaptic toxicity of Aβ, we employed a peptide inhibitor capable of effectively and specifically inhibiting the activities of all PAR-1/MARK family members. This inhibitor abrogated the toxic effects of Aβ oligomers on dendritic spines and synapses as assayed at the morphological and electrophysiological levels. Our results reveal a critical role for PAR-1/MARK kinases in AD pathogenesis and suggest PAR-1/MARK inhibitors as potential therapeutics for AD and possibly other tauopathies where aberrant tau hyperphosphorylation is involved

Pclo1980-2553 localizes to stress fibers induced by activated Daam1.

<p>Expression and immunostaining of COS7 cells transfected Myc tagged Daam1 isoforms (red) alone <b>(<i>A</i>)</b> or co-expresssed <b>(<i>B</i>)</b> with EGFP or EGFP-Pclo_1980-2553 (green). Alexa Fluor-conjugated phalloidin (blue) identifies actin rich structures including stress fibers. The scale bars are both 5 μm and apply to all images in the respective panels.</p

Pclo1980-2553 is targeted into filopodia when co-expressed with activated Daam1.

<p>(<b><i>A</i>)</b> Labeling of mitochondria with Mitotracker Green (upper panels, green in merge) in COS7 cells expressing ActA-mRFP or ActA-Pclo_1980-2553-mRFP (middle panels, red in merge) demonstrates localization of Pclo_1980-2553 sequences to the surface of mitochondria. <b>(<i>B</i>)</b> Expression of ActA-mRFP (top set of panels, red in merge) or ActA-Pclo_1980-2553-mRFP (bottom set of panels, red in merge) with Myc-C-Daam1 (green in merge) along with Actin labeling with Alexa fluor-coupled Phalloidin (blue in merge) reveals the accumulation of ActA-Pclo_1980-2553-mRFP labeled mitochondria in phalloidin positive filopodia. <b>(<i>C</i>)</b> Analysis of mitochondrial expression pattern indicates nearly all cells expressing ActA-Pclo_1980-2553-mRFP demonstrate accumulation in filopodia while this pattern is seen in less than half of the cells expressing ActA-mRFP. Data are expressed as mean with error bars representing standard deviation. For statistical analysis, comparison was made between the control cells (those expressing Mcy-C-Daam1 with ActA) and cells expressing Mcy-C-Daam1 with ActA coupled to Pclo_1980-2553 using a two-tailed t-test (* p<0.05).</p

Daam1 is a novel binding partner of Piccolo.

<p>(<b><i>A</i>)</b> Silver stained SDS PAGE gel of Piccolo antibody immunoprecipitation from P4 rat brain light membrane fraction reveals the presence of a 120 KDa band that was identified as Daam1 by mass spectrometry. <b>(<i>B</i>)</b> Western Blot analyses of similar SDS PAGE gels confirm the presence Daam1 in Piccolo immunoprecipitated fractions. Note that Bassoon, a known binding partner of Piccolo, is also immunoprecipitated while two other presynaptic proteins, Synapsin and Synaptophysin are not.</p

Daam1 interacts with the central region of Piccolo.

<p>(<b><i>A</i></b>) Schematic diagram of Piccolo depicting relative positions of different subdomains and composition of EGFP-tagged cDNA clones used in pull-down assays. <b>(<i>B</i>)</b> Western blot analysis of EGFP-tagged Piccolo fragments and Myc-Daam1 expressed and immunoprecipitated from COS7 cells with an antibody to GFP. (<b><i>C</i></b>) Schematic diagram of Daam1 depicting relative positions of different subdomains and organization of Myc-tagged cDNA clones. <b>(<i>D</i>)</b> Western blot analysis of Myc-tagged Daam1 constructs co-expressed with EGFP-Pclo_1980-2553 and immunoprecipitated from COS7 cells with an antibody to Piccolo. Q—polyQ domain; Zn1 and Zn2—zinc fingers 1 and 2; CC—coiled coil domain; PRS—proline rich sequence; PDZ—PDZ domain; C2—C2 domain; GBD—Rho GTPase binding domain; DID—Diaphanous Inhibitory Domain; FH1 and FH2—formin homology domains 1 and 2; DAD—Diaphanous Autoregulatory Domain.</p

Daam1 is present within the presynaptic bouton.

<p><b>(<i>A</i>)</b> Western blot analysis of fractions generated by differential centrifugation of adult brain lysate demonstrates the presence of Daam1 in synaptosomes (SYN) and to a lesser extent synaptic junctions (SJ). Signals with antibodies to known synaptic junctional components (Bassoon, Piccolo, CaMKII and PSD-95) versus a synaptic vesicle protein (Synaptophysin) demonstrate the integrity of the preparation. PNS—Post nuclear supernatant, S2- post hypotonic lysis supernatant, P2- post-hypotonic lysis pellet, SYN—synaptosomes, SJ—synaptic junctions. <b>(<i>B</i>)</b> Immmunostaining of cultured hippocampal neurons (16 DIV) with antibodies against Daam1 (green) and Piccolo (red) reveals a synaptic colocalization of the two endogenous proteins. Scale bar is 5 μm. <b>(<i>C</i>)</b> Axons from EGFP-Daam1 (green) expressing neuron (14 DIV, transfected at DIV 0) immunostained with antibodies against Piccolo (red) and MAP2 (blue) reveal EGFP-Daam1 puncta within Piccolo positive presynaptic boutons juxtaposed to MAP2 positive dendrites. Scale bar is 5 μm.</p