Date of Award
Doctor of Philosophy (PhD)
Alessandra d’Azzo, PhD
Matthew Ennis, PhD Elizabeth Fitzpatrick, PhD Gerard Grosveld, PhD Joseph Opferman, PhD
Alzheimer's Disease, Lysosomal Storage Disease, Microglia, Neuramindase 1, Neuroinflammation, Sialidosis
Background Sialic acids are key sugar moieties located at the non-reducing terminals of glycan chains on glycoproteins and glycolipids. By virtue of their location, they influence the functions and biochemical properties of the macromolecules they are bound to. Removal of sialic acids in mammalian cells is carried out by four sialidases, which are differentially expressed and localized in distinct subcellular compartments. Neuraminidase 1 (NEU1), the most abundant and ubiquitous of the four sialidases, functions primarily in the acidic environment of the lysosomes, but can hydrolyze substrates at the plasma membrane, at least in certain cell types. The enzyme initiates the sequential degradation of sialo-glycoconjugates by removing their terminal sialic acids. Through this activity, NEU1 modulates the sialic acid contents of its substrates, which affect their turnover rates, folding, ligand interactions, subcellular distribution, and subsequently the cellular pathways they control. Genetic deficiency of NEU1 leads to a pediatric neurodegenerative lysosomal storage disease known as sialidosis. In sialidosis, loss of NEU1 disrupts lysosomal catabolism, leading to accumulation of unprocessed substrates that drive pathogenesis by impinging on basic cellular processes, one of which is calcium-dependent lysosomal exocytosis. NEU1 negatively regulates this process by cleaving the sialic acids of LAMP1, thereby promoting its timely degradation. Since LAMP1 is required for the recruitment to and docking of lysosomes at the plasma membrane, loss of NEU1 results in an increased number of lysosomes preferentially docked at the plasma membrane ready to fuse and release their contents extracellularly. The end result is unrestrained and excessive lysosomal exocytosis from cells of different organs including the nervous system, with deleterious consequences for the integrity of plasma membranes and the extracellular matrix. These data from our laboratory established a paradigm, which we applied to investigate the course of neuropathogenesis in Neu1 KO mice, a faithful model of sialidosis. These mice develop a severe brain pathology sharing many hallmarks with Alzheimer’s disease (AD), including dystrophic neurites, progressive amyloidosis and neuroinflammation, features prominent in the hippocampal region. The amyloidogenic process starts with lysosomal accumulation of the amyloid precursor protein, a natural substrate of Neu1, which remains sialylated in absence of the enzyme and is abnormally cleaved into amyloidogenic peptides that are then released extracellularly via lysosomal exocytosis. These events initiate the formation of amyloid deposits in the Neu1 KO hippocampus and elicit a massive neuroinflammatory response mediated in part by the microglia. These findings have remarkable parallels with AD, where changes in the sialylation status of glycoproteins in both neurons and glia have been shown to correlate with disease stage in patients. In this dissertation we investigate microglia-mediated neuroinflammation and identify an AD- mimicking response occurring in Neu1 KO mice. Purpose We sought to determine the role of Neu1 in microglial functions by ascertaining whether microglia are activated and reacting in Neu1 KO hippocampi, assessing their phagocytic capacity, and investigating whether AAV-induced expression of human NEU1 in Neu1 KO mice would ameliorate microglial-mediated neuroinflammation. Results In the first section of the results, we showed that Neu1 KO microglia are higher in number, larger in size, and more granular when compared to wild-type cells. These features, combined with their amoeboid morphology, characterized Neu1 KO microglia as activated. Genetic analyses of Neu1 KO hippocampal tissue showed an upregulation of pro-inflammatory, myeloid-specific genes mimicking the immunoprofile of AD patients. We went on to demonstrate excessive lysosomal exocytosis in Neu1 KO microglia and established that these cells release increased levels of CCL3 and TNFalpha. Furthermore, we showed that lysosomal exocytosis contributes to the release of these signaling molecules by microglia. In the next section of the results, we established that Neu1 KO microglia have a reduced phagocytic capacity for multiple materials, the most relevant to AD being Abeta-42 oligomers. Through our analyses of microglial phagocytic receptors, we identified CD68 as a substrate of Neu1 and showed that Trem2 homeostasis is altered in Neu1 KO microglia. In these cells, Trem2 is abnormally sequestered into endo-lysosomal vesicles, wherein sialylated Trem2 accumulates in lysosomes, promoting excessive proteolytic cleavage of the protein into soluble Trem2 and Trem2-C-terminal fragment. Moreover, by comparing Neu1 KO and Neu1/Trem2 dKO microglia, we determined that Trem2 signaling in these cells contributes to the production of cytokines and chemokines, diminishes phagocytosis, and influences the levels of CD68 and Lamp1 at the plasma membrane. In the last section, we demonstrated that AAV-mediated replanishment of NEU1 ameliorates microglia-mediated neuroinflammation in Neu1 KO hippocampi. Microglia from AAV-treated hippocampi have reduced size, granularity, and expression of CD68. These cells also have lower levels of Lamp1 at the plasma membrane and secrete less CCL3 and TNFalpha, likely due to reduced lysosomal exocytosis. We determined that Trem2 cleavage is reduced in microglia from AAV-treated hippocampi, and phagocytic capacity is increased in a Trem2-dependent manner, likely due to corrected homeostasis of the receptor. Conclusions From our investigations we can conclude that Neu1 regulates microglial function by modulating lysosomal exocytosis, cytokine and chemokine production, and phagocytic capacity. As a negative regulator of lysosomal exocytosis, Neu1 can effectively reduce the levels of degradative hydrolases and neurotoxic signaling molecules that are released by microglia into the extracellular matrix. Neu1 acts as an antagonist towards the production of pro-inflammatory cytokines and chemokines, further reducing neurotoxic signaling by microglia. Neu1 promotes phagocytosis, enhancing the ability of microglia to clear harmful debris such as Abeta-42 oligomers, preventing subsequent neurodegeneration, and contributing to a neuroprotective microenvironment. Since all these processes occur in most microglia-mediated neuroinflammatory responses, including sialidosis and Alzheimer’s disease, and given that AAV-mediated replenishment of NEU1 effectively diminishes the on-going neuroinflammatory response in Neu1 KO mice, AAV-NEU1 gene therapy represents a possible therapeutic avenue for any neurodegenerative condition that possesses a NEU1-dependent pathogenic component.
Fremuth, Leigh Ellen (https://orcid.org/0000-0003-0408-9906), "Immunomodulatory Roles of the Lysosomal Sialidase Neuraminidase 1" (2022). Theses and Dissertations (ETD). Paper 586. http://dx.doi.org/10.21007/etd.cghs.2022.0593.
Available for download on Wednesday, May 17, 2023