| dc.description.abstracteng | In the adult brain, the extracellular matrix (ECM) forms lattices that sheath neurons and synapses. The exceptional longevity of ECM molecules lends these lattices a unique durability, and as such, they are deemed to stabilize neural circuits and restrict their plasticity. At the same time, the adult ECM retains the ability to be occasionally remodeled, in order to allow neural circuits to be altered throughout adulthood. According to the dominant paradigm in the current literature, this remodeling occurs through the transient release of proteolytic enzymes to cleave the ECM near synapses, followed by the secretion of newly-synthesized molecules that embed into the ECM, thereby resulting in ECM changes that fit this structure to the needs of synaptic plasticity. A problem arises when considering that structural changes to synapses in vivo are surprisingly frequent (on a timescale of minutes to hours). Supporting such frequent remodeling through de novo synthesis of ECM molecules would be costly for the cell in metabolic terms. Importantly, such a view is not in line with the measured lifetimes of these molecules, of weeks to months. How, then, can the cell sustain a continual remodeling of the ECM at synapses? In this thesis, I propose the existence of an additional mechanism, whereby the ECM can be continually remodeled through a recycling of its components. This mechanism lends the ECM the flexibility that is necessary for frequent synaptic changes. I expect that such a mechanism would operate constitutively, but would also be intimately linked to synaptic activity.
In Chapter 1, I summarize the existing knowledge on the configuration and function of the ECM in the adult brain, and discuss the potential interactions of ECM molecules with the pre- and postsynaptic machinery. After a brief discussion of the dominant paradigm for ECM remodeling through proteolytic cleavage, I present the body of literature to support the hypothesis of ECM recycling at synapses. Accordingly, I conclude that ECM molecules can be secreted in an activity-dependent manner from both the pre- and postsynaptic compartments. By contrast, I hypothesize that the locus of ECM entry into the recycling route is restricted to the postsynaptic side, where there is a considerably greater presence of local trafficking machinery. I remark, however, that it has not yet been established whether (and how) the presence of this local machinery is related to synaptic activity.
In Chapter 2, I present a collaborative project that endeavored to bridge the knowledge gap described in Chapter 1, and establish a link between the amounts of postsynaptic trafficking machinery and local synaptic activity. This was accomplished through the use of super-resolution microscopy and automated quantitative image analysis to successfully correlate the local distribution and abundance of several postsynaptic trafficking elements to local synaptic activity.
In Chapter 3, I present the primary investigation of this thesis, which directly tests the hypothesis of ECM recycling at synapses, using the glycoprotein TNR as an archetype. Here, we used an array of imaging assays, including super-resolution fluorescence imaging and nanoscale secondary ion mass spectrometry, to test the hypothesis that TNR molecules undergo continual recycling. We demonstrated the existence of a pool of mobile TNR molecules that are enriched at synapses, and cycle in and out of the perisynaptic ECM via a surprisingly long route (lasting approximately three days). Further investigation revealed that these molecules are trafficked as far as the Golgi apparatus in the neuronal soma, where they presumably undergo a re-glycosylation, and are then trafficked to synapses once again. Finally, we established a link between synaptic activity and the extent of TNR recycling.
Lastly, in Chapter 4, I discuss the primary conclusions of this thesis, as well as existing caveats and outstanding unanswered questions. I present possible experiments that could address these in the future, and discuss new areas of research that warrant further investigation. | de |