No description available.

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No description available.

No description available.

Autism spectrum disorder (ASD) encompasses a group of heterogeneous disorders that affect approximately 1% of children worldwide. ASD is characterized by two core symptoms, the first being deficits in social communication and interaction, and the second being restrictive and repetitive behaviours. Although environmental and genetic factors are known to contribute to the development of ASD, the etiology remains unknown. Genetic sequencing studies have implicated over 1000 genes with risk variants that are ASD-associated. Recent sequencing studies have highlighted that SCN2A, a gene that encodes the Voltage-Gated Sodium Channel Type II Alpha Subunit habours a large proportion of genetic risk variants for ASD. An emphasis was put on this gene because many of the top genes regulate transcription and cytoskeletal dynamics and not sodium flux aiding in regulating neuron excitability. Initial investigations of complete loss of Scn2a in mice led to perinatal lethality where heterozygous loss exhibited many behavioural phenotypes associated with ASD. Through our collaboration with Dr. Stephen Scherer (Hospital for Sick Children, Toronto) we identified two de novo truncating point variants in SCN2A. In our study, we focused on using human iPSC-derived neurons for disease modelling. We found these two variants caused a reduction in synapses suggesting that neuronal communication may be altered. Furthermore, electrophysiological characterization of the neurons harbouring the differing SCN2A variants showcased that loss-of-function (LoF) variants can produce differential phenotypes based on their location. Beyond the initial ion channel characterization, we wanted to probe whether cellular pathways were altered directly or indirectly by atypical neuronal activity. Proteomics of neurons expressing the more severe variant, p.R607*, found differentially expressed proteins (DEP)s that were upregulated and downregulated. Moreover, these DEPs were enriched and clustered into cellular pathways that were altered, with one of these clusters representing mitochondrial function. We functionally validated these findings in the same neurons and found corroboration between the molecular and cellular data of impaired mitochondria. Lastly, we used Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing to generate an isogenic model to validate our findings of the less severe p.G1744* variant. Together, this will aid in the discovery of new variant categorizations and targeted treatments for rescues of atypical neural connectivity or pathways that are altered downstream. / Thesis / Doctor of Philosophy (PhD)

Cone cyclic nucleotide-gated (CNG) channels are located in the retinal outer segments, mediating daylight color vision. The channel is a tetramer of A-type (CNGA3) and B-type (CNGB3) subunits. CNGA3 subunits are able to form homotetrameric channels, but CNGB3 exhibits channel function only when co-expressed with CNGA3. Mutations in the genes encoding these cone CNG subunits are associated with achromatopsia, an autosomal recessive genetic disorder which causes incomplete or complete loss of daylight and color vision. A missense mutation, aspartatic acid (Asp) to asparagine (Asn) at position 262 in the canine CNGB3 subunit (cB3-D262N), results in loss of cone function and therefore daylight blindness, highlighting the crucial role of this aspartic acid residue for proper channel biogenesis and/or function. Asp 262 is located in a conserved region of the second transmembrane segment containing three Asp residues designated the Tri-Asp motif. We exploit the conservation of these residues in CNGA3 subunits to examine the motif using a combination of experimental and computational approaches. Mutations of these conserved Asp residues result in a loss of nucleotide-activated currents and mislocalization in heterologous expression. Co-expressing CNGB3 Tri-Asp mutants with wild type CNGA3 results in functional channels, however, their electrophysiological characterization matches the properties of homomeric CNGA3 tetramers. This failure to record heteromeric currents implies that Asp/Asn mutations impact negatively both CNGA3 and CNGB3 subunits. A homology model of canine CNGA3 relaxed in a membrane using molecular dynamics simulations suggests that the Tri-Asp motif is involved in non-specific salt bridge pairings with positive residues of S3 - S4. We propose that the CNGB3-D262N mutation in daylight blind dogs results in the loss of these interactions and leads to an alteration of the electrostatic equilibrium in the S1 - S4 bundle. Because residues analogous to Tri-Asp residues in the voltage-gated Shaker K+ channel superfamily were implicated in monomer folding, we hypothesize that destabilizing these electrostatic interactions might impair the monomer folding state in D262N mutant CNG channels during biogenesis. Another missesnse sense mutation, Arginine (Arg) to tryptophan (Trp) at position 424 in the canine CNGA3 subunit (cA3-R424W), also results in loss of cone function. An amino acid sequence alignment with Shaker K+ channel superfamily indicates that this R424 residue is located in the C-terminal end of the sixth transmembrane segment. A3-R424W mutant channels resulted in no cyclic nucleotide-activated currents and mislocalization with intracellular aggregates. However, the localization of cA3-R424W mutant channels was not affected as severely as the Asp/Asn mutation in S2 Tri-Asp motif, showing a lot of cells with the proper localization of Golgi-like and membrane fluorescence. Moreover, the substitution of Arg 424 to Lysine (Lys), conserving the positive charge, preserved channel function in some cells, which is different from the results of the S2 Tri-Asp motif in which the Asp/Glu substitutions, conserving the negative charge, leads to loss of cyclic nucleotide-activated currents. Even though these missense mutations are both associated with canine daylight blindness, the Arg 424 residue might not be as critical for folding as the Tri-Asp residues in the S2 Tri-Asp motif and might be more of a problem in channel structure and function. The cA3 model relaxed with MD simulations indicated a possible interaction of Arg 424 with the Glu 304 residue in the S4-S5 linker. This hypothesis is supported by electrophysiological data in which the double mutation of reversing these residues, Glu 306 to Arg and Arg 424 to Glu (E306R-R424E) preserves channel function. In the model, this salt bridge appears to contribute to stabilization of the open pore state. The R424W mutation might disrupt the salt bridge formation, leading to deforming and closing the pore region. / Biology

The large conductance calcium dependent potassium (BKCa) channels are expressed in a large variety of cell types including neurons where they modulate excitability and action potential shape. Within the enteric nervous system, stretch-sensitive BKCa channels are expressed on intrinsic primary afferent neurons (IPANs) where they decrease the neurons’ excitability during intestinal contractions. A major determinant of peristalsis is slow excitatory neurotransmission (sEPSPs) within the IPAN to IPAN sensory network, and we wondered whether such transmission might also alter BKCa channel opening. All experiments were performed on longitudinal-muscle myenteric preparations prepared from jejunal segment taken from freshly euthanized adult male Swiss Webster mice. With the myenteric plexus exposed by microdissection, BKCa channel activity was recorded in cell-attached mode via the patch clamp technique. BKCa channel activity was recorded before and after presynaptic electrical stimulation, which was designed to evoke postsynaptic sEPSPs. The morphotype was verified by intracellular injection of a marker dye (neurobiotin). In addition, a blocker and opener were used to identify the effects of BKCa currents on IPAN properties. Analysis of unitary channel recordings revealed increased BKCa open probability (NPo) at fixed trans-patch potentials following sEPSPs. All BKCa channels were independently voltage sensitive with increased NPo during patch depolarisation. Analysis of whole-cell experiments also revealed BKCa channels have a significant effect on the undershoot amplitude of action potentials, and the rate at which IPANs repolarise. This study demonstrates that sEPSPs within the enteric nervous system modulate the function of BKCa channels in IPANs adding to the mechanistic understanding of enteric synaptic transmission and providing a potential target for therapeutic modulation of enteric nervous system excitability. / Thesis / Master of Science (MSc)

Thesis / Master of Science (MS)

New abstract (saved as much of your structure/wording as possible): In mammals, sound localization along the azimuth is computed in part in the lateral superior olive (LSO), a binaural nucleus in the brainstem. Information about the location of the sound source is derived from differences in sound intensity at the two ears, the Interaural Level Difference (ILD). Within each LSO, principal cells compute ILDs by integrating an excitatory input carrying intensity information from the ipsilateral ear with an inhibitory input carrying intensity information from the contralateral ear. This computation requires that the phenotypically distinct inputs onto individual LSO cells be matched for sound frequency. The process of ‘aligning’ and refining the inputs for frequency information occurs during the first few postnatal weeks in rats, through modifications of synapse strength and cell morphology. Our lab studies the distribution, and re-distribution, of these converging inputs during the early period of circuit refinement. A common strategy for examining spatial distribution of synapses is through anatomical techniques, including for example immunohistological methods for localizing specific synaptic proteins. Ultimately, however, we need to understand how synapse position affects the functional response. Asking this kind of question requires the ability to stimulate individual synapses while recording from dendrite or cell body, an approach for which we use laser scanning photostimulation (LSPS). I designed two LSPS systems in order to stimulate the post-synaptic sites of excitatory or inhibitory inputs on LSO principal neurons while recording at the cell body using whole-cell patch clamp. I researched many optical designs and technologies when fine-tuning my design. My designs and initial groundwork will help a future lab member finish one or both of the LSPS designs. / Thesis / Master of Science (MSc)

No description available.