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'Effect of Disease-Causing Mutations of the Alkali Cation/Proton Exchanger on Neuronal Morphology and Function' Undergraduate Research Project Application Form.

INSTRUCTIONS - PROFESSORS: Fill out Sections A & B then submit this form online. (You will receive an email copy of the form. The Office for Undergraduate Research in Science will also post the project online, indicating whether the project is open for students to apply or taken.) DONE
INSTRUCTIONS - STUDENTS: You may receive this form by email, or you may download it after it has been posted. Either way, print this form. Complete and sign Section C on the hardcopy. Ask your supervisor to sign Section D. Take it to the department corresponding to the course number in Section A (this may or may not be your own department). Do not register for a '396' course on Minerva until you receive departmental permission. Have a discussion with your supervisor about time/work expectations, keeping in mind that this is a 3-credit course (roughly, 10 hours per week for 12 weeks). Remember that a '396' course is an elective.
INSTRUCTIONS - DEPARTMENTS: After the unit chair/director/designate approves (or not) this project, notify student. If approved, please give student permission to register on Minerva, and fax this form (with signatures) to the Office for Undergraduate Research in Science.
QUESTIONS OR FEEDBACK? Contact Victor Chisholm by email, or phone 514-398-5964.
Name: John Orlowski
Email: john [dot] orlowski [at] mcgill [dot] ca (john dot orlowski at mcgill dot ca)
Supervisor's department: Physiology
Supervisor's department (if none of the above)  
Course number: PHGY396 (Physiology)
Term: Winter 2010-2011
Project start date: January 4, 2011
Project end date: April 8, 2011
Project title: Effect of Disease-Causing Mutations of the Alkali Cation/Proton Exchanger on Neuronal Morphology and Function
Project description:

Recent genetic studies have identified a three-amino acid deletion mutation (Δ338WST340) in a pH-regulating transporter, the solute carrier Na+/H+ exchanger isoform 6 (SLC9A6/NHE6), that is linked to the development of a severe form of mental retardation characterized by the virtual absence of speech, autism spectrum disorder, epilepsy, late-onset ataxia, weakness and dystonia. NHE6 is located in recycling endosomes of many cell types and is thought to be involved in the regulation of vesicular luminal pH and trafficking. In neurons, it is especially abundant in postsynaptic recycling vesicles of dendrites and dendritic spines. However, the molecular and cellular mechanisms whereby mutations in NHE6 lead to neuronal dysfunction are unknown. Since morphological examination of individuals with mental retardation and autistic disorders often display abnormalities in synapse structure, this project will test the hypothesis that the disease-causing mutation in NHE6 will disrupt the proper trafficking of NHE6-containing endosomes that are normally required for optimal synapse formation and function.

For this project, dissociated embryonic hippocampal neurons will be prepared from transgenic mice lines expressing the myristoylated green fluorescent protein (mGFP) under the Thy 1 promoter unless otherwise stated. The mice were constructed by a collaborator Dr. P. Caroni (FMI, Basle, Switzerland). The major advantage of using mGFP preparation is that one obtains a much higher level of resolution of the neuronal morphology compared to cytosolic dye injection. The aims of this project are:

  1. To characterize the subcellular distribution of wild-type and mutant NHE6 transfected into in primary cultures of dissociated hippocampal neurons. This will be accomplished by constructing chimeric genes containing wild-type (WT) and mutant (Δ338WST340) NHE6 fused to the fluorescent protein mCherry (mChFP) and transfecting these constructs into primary cultures of dissociated embryonic hippocampal neurons. To precisely define the nature of the NHE6-containing vesicles, dual-labelling experiments will be performed at the light microscopy level using well characterized, commercially-available, antibodies that recognize known markers of dendrites (microtubule-associated protein 2), axons (anti-neurofilament-H), early endosomes (e.g., Rab5), recycling endosomes (e.g., Tf-R), large dense-core vesicles (e.g., synaptotagmin) and synaptic vesicles (e.g., synaptophysin). The colocalization of NHE6 with ionotropic (AMPA-R, NMDA-R and Kainate) glutamate receptors will also be examined, as all of these receptors are known to enter recycling endosomes and are involved in learning, memory and other physiological behaviours.
  2. To determine if NHE6 is necessary and/or sufficient for synapse formation and maintenance. Based on our preliminary localization results, morphometric analyses will be performed to determine the effects of overexpression of mutant NHE6-ΔWST-mChFP on dendritic spine formation using dynamic imaging. The number and shape of dendritic spines will be quantified and compared to control non-transfected or NHE6-WT-mChFP transfected sister cultures.
Prerequisite: 1 term completed at McGill + CGPA of 3.0 or higher; or permission of instructor.
Other prerequisite, if applicable:  
Grading scheme (The final report must be worth at least 50% of final grade): Final grade shall be based on laboratory performance as evaluated by the research supervisor (50%) and the final written research report (minimum 10 pages) graded by the supervisor and the course coordinator or the coordinator's delegate (50%).
Other project information:
Project status - This project is: Taken. The professor has no more '396' projects this term.
How students can apply: N/A; this project is filled.
If other, please specify:  
Ethics, safety, and training
Which of the following, if any, is involved? One or more of the following
Animal subjects [x]
Human subjects [ ]
Biohazardous substances [x]
Radioactive materials [ ]
Handling chemicals [ ]
Using lasers [x]
Supervisors are responsible for the ethics and safety compliance of undergraduate students.
Do not complete this section unless/until the student is identified.
McGill ID:  
Email (first [dot] last [at] mail [dot] mcgill [dot] ca):  
Program (E.g., B.Sc. Maj. Chem. Min. Biol.):  
Level (U0 / U1 / U2 / U3):  
Student signature - I have not applied for another '396' course in this term:  
Do not complete this section unless/until the student is identified.
Supervisor: I give my permission for the student identified in section C to register for this project under my supervision.
Supervisor's signature:  
Unit chair/director/designate: I certify that this project conforms to departmental requirements for 396 courses.
Unit chair/director/designate's name:  
Unit chair/director/designate's signature: