Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)


Biomedical Sciences



Research Advisor

Alessandra d’Azzo, Ph.D.


Max L. Fletcher, Ph.D.; Detlef H. Heck, Ph.D.; Camenzind G. Robinson, Ph.D.; Lindsay A. Schwarz, Ph.D.


Beta-Galactosidase; Enzyme Replacement Therapy; ER-PM Junctions; GM1-Gangliosidosis; Lysosomal Storage Diseases; Membrane Contact Sites


Background. GM1-ganglisidosis is a rare neurodegenerative lysosomal storage disease caused by the deficiency of the lysosomal enzyme β-GAL, resulting in the accumulation of its target substrate GM1. GM1, a glycosphingolipid found primarily in the PM of neurons, is known to modulate Ca2+ flux through its interactions with Ca2+ channels and Ca2+ binding proteins. GM1 is also known to promote neuritogenesis through the activation of phosphorylated TRKs, receptors for neurotrophic factors. Under disease conditions, GM1 accumulates not only in the PM but also in the intracellular membranes of organelles, altering their membrane composition and functions. Organelles are dynamic, constantly interacting with each other and the PM through specialized membrane microdomains, known as membrane contact sites (MCS). MCS have distinct protein and lipid composition and facilitate the transfer of lipids, proteins, metabolites and ions, such as Ca2+, between the organelles via non-vesicular transport. The two most studied MCS are those formed between the ER and mitochondria, known as mitochondrial-associated ER microdomains, or MAM, and those between the ER and PM, known as ER-PM junctions. These MCS are important for controlling Ca2+ homeostasis, through Ca2+ transporters and channels, and have been extensively studied for their role in phospholipid and cholesterol transport. These two lipids, along with glycosphingolipids, are the major lipid constituents of membranes; however, very little is known about the role of glycosphingolipids in MCS dynamics. Using a mouse model deficient for the lysosomal enzyme β-Gal, we have been able to study the role of GM1 in MCS formation and functions. GM1 accumulation was shown to increase the number of MAM tethering events. We have also shown that GM1 accumulates at the MAM triggering a phosphorylated IP3R-mediated ER Ca2+ depletion, resulting in the activation of the unfolded protein response. This efflux of Ca2+ is then buffered by the mitochondria, through the MAM, resulting in mitochondrial Ca2+ overload, activation of the caspase cascade and neuronal apoptosis. Purpose. Given the similar functions between the MAM and ER-PM junctions in Ca2+ dynamics and lipid transport, we sought to determine if GM1 accumulation at the respective membranes of β-Gal KO neurons would facilitate the formation of these MCS and alter the functions of the ER-PM junctions with respect to changes in Ca2+ regulated processes. We also aimed to test the efficacy of an ERT approach for ameliorating some of the neurodegenerative aspects of the GM1-gangliosidosis mice, using an RTB-conjugated recombinant β-Gal that has the ability to cross the blood brain barrier. Results. In the first set of results, we used TEM to demonstrate that β-Gal KO neurons have a significantly higher number of ER-PM junctions than WT neurons. This GM1-mediated phenomenon was also apparent in β-Gal KO primary neurons as well as GM1-l

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.




2022-018-Weesner-DOA.pdf (199 kB)
Declaration of Authorship

Available for download on Tuesday, November 07, 2023