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Targeted knockdown of murine Prickle1 gene to document its role in human Progressive Myoclonic Epilepsy.

Targeted knockdown of murine Prickle1 gene to document its role in human Progressive Myoclonic Epilepsy.

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  • 1. INTRODUCTIONMutant variants of the Prickle1 gene in humans have been shown to give rise to a previouslyundocumented subtype of Progressive Myoclonus Epilepsy, a neurological disease characterizedby seizures and ataxia. Encoding proteins essential for polar cell planarity and transcriptionregulation in neural cells, the Prickle1 gene is present as a homolog in the laboratory mouse.Systematic genetic studies performed on laboratory mice can be used to investigate the gene’srole in disease causation, withRNAi-mediated gene ablation and restoration coinciding with thepresence and absence of symptoms respectively. Cre-Lox technology was additionally employedto maintain spatial and temporal disease manifestation.HYPOTHESISThe Prickle1 homolog in mice plays a significant role in differentiation, cell fate determination,and cell signaling, with its ablation results in the development of a myoclonic epilepsy syndromesimilar to that seen in PME-positive humans. Restoration of gene function should likewisealleviate symptoms. In vitro gene knockdown via RNA interference is analogous to the loss-of-function observed in diseased individuals, and its functional ablation at different time points andin different tissues should produce a pattern of characteristic symptoms that provide valuableinformation about the disease.AIMS: - To investigate the effects of mutant Prickle1 proteins in mouse models such that more can be elucidated regarding the syntenic human disease of Progressive Myoclonus Epilepsy - To investigate the severity and symptoms of the disease under spatial control - To investigate the severity and symptoms of the disease under temporal controlBACKGROUND AND SIGNIFICANCEA new subtype of Progressive Myoclonus Epilepsy (PME) has been documented in scientificliterature, with linkage analysis revealing no participation of known epilepsy genes. The diseaseis familial, highly penetrant, and progressive, manifesting in childhood and characterized byfrequent, recurring seizures (synchronous spikes of abnormal brain activity). Ataxia, which isdysfunctional muscle control, is generally observed and considered a hallmark sign, with patientspresent with unstable gait and coordination difficulties. While a normal lifespan provesachievable, a significant proportion of patients do not reach adulthood, due primarily to theaffected muscles impeding the essential processes of respiration and swallowing.Displaying autosomal recessive inheritance patterns, the prevalence of PME is yet unknown,though its high incidence in large, consanguineous Middle-Eastern families suggests a potentialfounder effect caused by novel mutations in a single ancestral allele. Genetic analysis reveals a
  • 2. transitional missense mutation in the Prickle1 gene mapped to human chromosome 15, withcorresponding orthologs found in Drosophila and Mice. The characteristic PET domain is largelyinvariant between species, suggesting functional importance, and the common missense variantR104Q observed within the PET domain of diseased individuals is thus thought responsible forfunctional ablation and disease causation. Other Prickle1 variants are less commonlydocumented, if at all.The native Prickle1 gene performs a variety of functions, encoding five documented proteinisoforms. The 883 amino acid long Prickle1 protein is integral to the non-canonical Wntsignaling pathway, participating in the biochemical cascade that, in conjunction with otherknown signaling pathways, leads to planar cell polarity determination and associated neural cellpatterning. This includes neural tube closure and the regulated formation of the rudimentaryspinal cord, in addition to directed organ positioning with conventional asymmetry. The non-viability of mice nullizygous for Prickle1 suggests its importance in early developmentaldifferentiation, though this embryonic lethality is not documented in human individuals.Histopathological analyses of spontaneously aborted mouse embryos display chaotic cellularpositioning with skewed apical-basal orientation, with indistinct and improper segregation andmigration of the three germinal layers of the fledgling inner cell mass. The compromisedstructural integrity is understandably fatal.Prickle1 proteins are also suspected to work in coordination with the RE1-SilencingTranscription Factor (REST), which is responsible for the transcriptional downregulation ofneuronal genes including those encoding neurotransmitters or receptors. Rigid regulation ofdownstream genes is essential for the maintenance of coordinated neuromuscular function thatallows for the fine motor skills required in human kinesiology, and Prickle1 protein complexeswith REST, allosterically impeding its translocation to the cellular nucleus whereupon it canconstitutively suppress neuronal gene function. Conversely, ablation of Prickle1 protein leads toectopic and inappropriate REST-induced neuronal gene silencing, which may be responsible forthe ataxia/seizure phenotype observed in diseased individuals. Co-immunoprecipation hasrevealed independent REST proteins in PME patients with a consequent downward shift on aWestern blot assay, suggesting that R104Q mutant Prickle1 protein is unable to bind to RESTand regulate its function.Suspected secondary functions include encoding nuclear receptors specific for stereospecificligands, possibly with accessory zinc ions, and regulation of nuclear import, export, andpermeability.Immunostaining with rabbit polyclonal antibodies revealed that Prickle1 proteins are expressedwithin brain tissue, primarily within the hippocampus, thalamus, cerebellum, and cerebral cortex.The expression is not extended to glia, though it is suggested that differential expression patternsare concordant with developmental stage. The cerebellum’s indispensability for normal posturingand balance strongly suggests that ataxia is the result of its abnormal function via mutant
  • 3. Prickle1. Epilepsy syndromes have described dysfunctions in the other cerebral regions ascausative.Elucidating the function of Prickle1 proteins and their behavior in both wildtype and mutatedforms can lead to the development of directed cures. As such, mouse models are employed forinvestigation. Homozygous mutant mice are postulated to develop the same debilitatingsymptoms as their human counterparts. Spatial expression of mutant genes allow for thelocalization of affected tissues based on symptom quantification, and temporal expression ofmutant genes allows for the distinction of PME as either an inborn neurogenerative or anacquired syndrome which may present only in later life.The evolutionarily-conserved RNAi phenomenon is a highly-regulated form of endogenous,post-transcriptional gene regulation. Processed, single-stranded RNA strands of intermediatelength (commonly 21-24 nucleotides) bind complementarily to nascent mRNA transcripts,resulting in their systematic degradation via the catalytic components of the multiprotein RNSInduced Silencing Complex (RISC) the former is incorporated within. The principle of antisenseRNA intervention can thus be replicated in vitro, facilitating the targeted knockdown of specificgenes. This abortive mechanism inhibits the downstream protein synthesis and processing,effectively suppressing the gene’s inherent function.Derived from DNA expression vectors, laboratory-designed shRNAs comprise antisense andsense regions, making them recognizable substrates of the native murine DICER catalystresponsible for their enzymatic introduction into a functional RISC complex. While concordantwith the delivery method, silencing rates of 90% have been documented, as quantified byhaplosufficiency assays.While thus effective in gene ablation, in vitro RNAi is known for producing undesirable ectopiceffects. It is thus utilized in tandem with conventional Cre-Lox recombination technology thatprovides a measure of spatial and temporal control.The bacterial-derived Crerecombinase proteinis capable of causing recombination events when exposed to DNA fragments bounded bycorresponding loxP sites. This recombination can be an inversion, translocation, or deletion,depending on the orientation of the loxP sites, resulting in the formation of a stable Hollidayjunction and the desired translocation event, after which endogenous DNA ligase catalysesstrand re-joining.Thus, the introduction of specific mutations leading to disease development theoretically impliesthat converse removal results in corresponding disease resolution. This hints at a potential cure,though further scientific research is imperative before proper, significant conclusions can bemade.EXPERIMENTAL DESIGNSection 1– Introducing the R104Q mutation
  • 4. Recessive diseases are generally characterized by loss-of-function mutations. However, it isimperative to distinguish between homozygous mutants and the nullizygote. The viability ofhuman homozygotes indicates that Prickle1 function is still present, albeit at drastically reducedlevels. Nullizygote is therefore non-synonymous with homozygous mutant.Fistly, we capitalize upon the RNAi pathway native to mice, in which experimentally-designedshRNA hybridizes to complementary native sequences which are then systematicallydegraded by the catalytic Argonaute action of the RISC complex the siRNAs are incorporatedinto. Controlled by a proximal, ubiquitous UG promoter, the complementary nucleotide sequencelies upstream of a Puromycin-resistance reporter. Mimicking murine microRNA, it is processedby endogenous Dicer and Drosha enzymes and loaded into the cytoplasmic RISC complex.Perfect complementarity results in enzymatic degradation of the immature Prickle1 transcript,while imperfect complementarity leads to suppression of the RISC complex, reducing geneexpression in either scenario. This is particularly important, affording a measure of toleranceagainst potential SNPs resulting from the degeneracy of the genetic code.Figure 1.1.RNAi-mediated gene regulation/suppressionWhile commercial shRNAs can be bought via Origene, manual designing is possible so long asthe sequence of the desired knockout is known. shRNA presents a characteristic hairpin loop
  • 5. structure achieved via self-complementation, and it is these sense and antisense regions that arecapable of hybridizing to the destined knockdown gene.Figure 1.2. The characteristic shRNA construct used for in vitro gene knockdowns. Restrictionsites are required for introduction into recipient plasmids or viral vectors.Figure 1.3.Prickle1-targetted shRNA (Origene), with a Puromycin resistant marker used inselection for Lentiviral plasmids that have incorporated the construct via electrophoretictransfection methods.(Manual synthesis: Native U6 promoters contain essential GGG sites from which transcription isinitiated, and the Guanine repeats within the target gene sequence are thus considered suitablestart sites. Directly distal would be the selected sense sequence and the following reversed sensesite, with an intervening central 8-nucleotide loop containing a bacterial restriction enzyme site.A second, complementary oligonucleotide sequence of similar configuration then hybridizes tothis strand, forming a double-stranded DNA fragment with overhangs specific to their internalrestriction enzyme recognition sites, the latter used to facilitate the fragment’s introduction intoviral or plasmid vectors.)After integration of construct-carrying plasmids, Lentivirus-associated delivery was performeddue to its non-episomal nature. Unlike conventional Adenoviruses which do not integrate intotranscriptionally-relaxed euchromatin, Lentivirus show successful incorporation and thusperpetuate through the progeny. Circumvention of random, potentially deleterious mutagenesis
  • 6. was achieved by using an integrase-deficient lentivirus,Intranasal introduction was favoured asan administration route as viruses show functional optimization in the lower temperatures of therespiratory tract as opposed to the slightly-warmer temperature of blood.Assuming sucessful gene knockdown, the mice should now exhibit the classical symptoms ofPME. 0.5cm of their tails was removed, and identification ofG(0) transgenic founder mice wasperformed via qPCR with primers specific for the U6 promoter and Puromycin-resistant sites (5-CGAAGTTATCTAGAGTCGAC-3, and 5-AAACAAGGCTTTTCTCCAAGG-3 respectively).While mutant mice are expected to experience seizures, seizure incidence is unpredictable.Injected administration of anxiogenicPentylenetetrazol is thus used to induce seizures in bothaffected and control mice, with seizure susceptibility, intensity, duration, and recovery timebeing documented. Simultaneous EEG monitoring is used to quantify abnormal brain activity,while functional Magnetic Resonance Imaging can be employed to determine which cerebralregions are active during seizure occurrence.As ataxia is a hallmark symptom of PME, mice are subject to a battery of kinaesthetic tests. Theability to balance on a horizontally-rotating rotarod and the presence/absence of tremors canreveal functional disability in mouse motor coordination. Electronic gait analysis can be used todocument mouse walking stability and speed, both of which are believed to be compromised inthe ataxic mouse. General mouse behaviour is also documented, elucidating any secondary issuesthat may be co-morbid with PME (e.g. below-average height/weight).Section 2 – Conditional Mutagenesis (Cre-Lox Recombination)RNAi usage circumvents embryonic lethality issues as shRNAs can be introduced at any age.However, traditional Cre-Lox recombination provides an unrivalled measure of spatial andtemporal control. In the former, Cre is expressed only in a particular cell line, driven by aspecific promoter, and the corresponding translocation events will be exclusive to that cell linewhen crossed with a mouse presenting specific LoxP junctions. In the latter, inducible agentsincluding tetracycline derivatives can be employed to catalyse time-specific translocation eventsThus, Cre-Lox mediated shRNA targeting the Prickle1 gene has been developed.Here, a floxedStop cassette containing terminating thymidine repeatswas inserted within the U6promoter driving shRNA expression such that RNAi performance is inhibited. The vectorconstruct was introduced via direct pronuclear injection, and crossing this floxed mouse with oneexpressing Cre-recombinase would allow for the effective deletion of the intervening Neocassette, restoring U6 promoter function and the downstream RNAi-mediated silencing of thePrickle1 gene product.Crossing the floxed specimens with mice expressing Cre in different tissues allowed for theelucidation of Prickle1’s role in the respective tissues.
  • 7. 406-week-old C57BL6 mice were thus purchased from GENSAT. - 10 of these mice were STOCK Tg(Dbp-cre)MN120Gsat/Mmucd (expression in Cerebral Cortex, Thalamus, Cerebellum, Hippocampus) - 10 of these mice were STOCK Tg(Ascl1-cre)ND216Gsat/Mmucd(expression in the Hippocampus) - 10 of these mice were STOCK Tg(Dlx5-cre)MO36Gsat/Mmucd (expression in the Thalamus) - 10 of these mice were STOCK Tg(Cxcl11-cre)KN257Gsat/Mmucd (expression in the Cerebellum)Figure 2.1. : A floxed STOP cassette introduced in a mouse prevents U6-driven shRNAproduction. Removal via Cre restores this.Floxed mice are then observed and subjected to the same behavioural tests mentioned in Section1.1.Gene ablation in different brain regions should lead to the manifestation of symptomscorresponding to the region in question. Fluorescence signals are observed in respective braintissues under microscopy. 5 mice from each of the Cre-reporter lines are euthanized via cervicaldislocation, with transverse sections of brain tissue being excised and isolated. The sections arehomogenized and subjected to a classical Western blot procedure with protein presence andmolecular weight being quantified via polyacrylamide gel electrophoresis. This is compared witha set of control mice.Prickle1 protein is said to bind to the REST protein in vivo, and co-immunoprecipitation ofREST in mutant mice and controls will reveal the presence or absence of such allostericcoordination.Section 3- Inducible Cre-lox systems
  • 8. The tetracycline-derivate of Doxycycline is used for the time-specific induction of Prickle1-tagetted shRNA function. In this model, a tetR/O shRNA vector is employed, preventing theconstitutive expression of the shRNA that inactivates the Prickle1 gene via RNAi knockdown. Inthe absence of the inducer, Doxycycline, the Tetracycline repressor binds constitutively to itscorresponding operator, disabling it. Both the repressor and the operator are part of theinsertional plasmid construct, the latter driven by a modified H1 promoter, with the formercontrolled by a codon (CAG) one.Figure 3.1: The introduction of Doxycycline-sensitive TetO and TetR elements can regulateproduction of the shRNAs within the same construct.Figure 3.2:Detailed manual synthesis of the inducible construct, requiring specific restrictionsites for incorporation into recipient plasmids/ vectors.Doxycyline is introduced via intraperitoneal injection, whereupon it binds to the Tet repressor,allosterically modifying its physical configuration such that a loss of necessary stereospecificityis lost. This results in the physical inability to complex with the Tet operator just upstream of theshRNA construct, restoring the former’s operative function. shRNAs are once more produced,knocking down Prickle1 gene expression wherever expressed.
  • 9. The mice are then observed and subjected to the same behavioural tests mentioned in Section 1.1.Of interest to me is the investigation of PME as a developmental disorder or one that occurs inthe functioning adult brain. As Doxycycline injections allow for the temporal control oversymptom manifestation, different mice were given Doxycycline at different time points of theirlife i.e. at 6-, 8-, 10-, 12- weeks of age and observed with regard to symptoms and behaviouraltests. Different degrees of affectedness would determine the disease classification i.e.development of the disease within 6 weeks but not after that would indicate aneurodevelopmental disorder.HYPOTHESIZED RESULTSMice heterozygous for the Prickle1 mutation are likely asymptomatic, though haploinsufficiencymay be observed via protein quantification. Mice that undergo a loss of heterozygosity willdevelop symptoms based on the cerebral region that the mutation was introduced in.Table 4.1Brain Region Symptoms observedHippocampus Seizures, with hippocampal sclerosis observed upon autopsyThalamus Generalized tonic-clonic seizuresCerebral Cortex Focal or absence seizuresCerebellum AtaxiaThe same results are likely to be observed in floxed Mice (inducible or not).Behavioral tests should show vastly different behaviour between mutants and controls.Table 4.2.Behavioral test Control Mice Prickle1-knockout miceRotarod Analysis Balancing ability intact Balancing ability compromisedTremor Analysis No tremor observed Tremor of varying intensityTail Suspension Test Relaxed phenotype Clasping phenotypeGait Analysis Predictable footprints Erratic footprintsMouse Maze Performed quickly Performed slowlyIt is worth noting that the discontinuation of Doxycycline treatment will possibly result in areversal of symptoms due to the impermanent nature of that inducible system. The restoration ofa normal phenotype may suggest that a complete cure is possible.MATERIALS AND METHODS
  • 10. Subjects: Healthy, 6 week old mice were obtained from the Jackson and GENSAT laboratoriesand they were housed and euthanized according to guidelines meeting ethical criteria. An equalnumber of male and female mice were obtained. Approximately half of the starter mice wereC57BL6 mice and the other half were Agouti, allowing for the identification of heterozygotesand homozygotes via characteristic coat colour inheritance patterns.Immunostaining protocols (Western Blots):Blocking agent was 3% Bovine Serum Albumin. 0.5 micrograms/ml. Anti-prickle1 rabbitpolyclonal antibody was used. Secondary mouse anti-Prickle1 antibody was conjugated to afluorescent protein, producing a quantifiable signal which could be observed and analysed.Protocol: Excised brain tissue segments were homogenized and treated with Sodium DodecylSulfate to confer upon them universal negative charges such that migration is determined only bymolecular weight. Samples are loaded into 5% Polyacrylamide gels and subjected to voltageexcitation in two dimensions, the first measuring isoelectric point, the other measuring molecularweight.The result is electroblotted onto Niitrocellulose membranes then probed with antibodies in asequential function. Mutant mice should show no or little Prickle1 protein product as comparedto control mice, which should show a Prickle1 protein profile similar to that documented fromknown protein databases.Polymerase Chain ReactionSuccessful lentiviral-mediated construct uptake should reveal characteristic complementarynucleotides upon PCR amplification. 5-CGAAGTTATCTAGAGTCGAC-3, and 5-AAACAAGGCTTTTCTCCAAGG-3 primers were designed targeting the flanking U6 andPuromycin sites, allowing for mass amplification of the shRNA construct (if present). Thepresence of the construct would reveal a band (as made observable by treatment agent EthidiumBromide) upon subsequent polyacrylamide electrophoretic assaying; this band is not observed incontrol mice.Possible Limitations: RISC-oversaturation, Lentiviral infection, Lentiviral-degradation viamurine immunity, varying shRNA expression levels due to integration sites/copy number.Reference list: 1. A Homozygous Mutation in Human PRICKLE1 Causes an Autosomal-Recessive Progressive Myoclonus Epilepsy-Ataxia Syndrome, Alexander G. Bassuk,1,2,3 Robyn H. Wallace,7 Aimee Buhr, 2. RNAi-based conditional gene knockdown in mice using a U6 promoter driven vector, VivekShukla, Xavier Coumoul#, Chu-Xia Deng, Int J BiolSci2007; 3(2):91-99. doi:10.7150/ijbs.3.91
  • 11. 3. Cre-lox-regulated conditional RNA interference from transgenes, Andrea Ventura, Alexander Meissner, Christopher P. Dillon Department of Biology, Center for Cancer Research, and Howard Hughes Medical Institute and §McGovern Institute, Massachusetts Institute ofTechnology,4. Conditional RNAi in miceAljoschaKleinhammeraJan Deussingc5. A transgenic approach for RNA interference-based genetic screening in mice, ShaohuaPeng , J. Philippe York, and Pumin Zhang6. Taconic: Discover the Power of RNAiin vivo! Innovative RNAi gene knock down technology for target validation,gene function analysis, and disease modeling. THANK YOU FOR READING!!! 