Prion diseases are sporadic and infectious neurodegenerative disorders caused by PrPSc, a misfolded and aggregated isoform of the cellular prion protein, PrPC. PrPSc accumulates and spreads through the CNS, causing neurodegeneration, gliosis, and ultimately death. However, the mechanisms underlying prion transport and pathogenesis remain poorly defined. The goals of my thesis research are to uncover properties of both prions and cellular pathways that facilitate prion spread into the central nervous system (CNS). First, we analyzed the biochemical properties of prion strains that enable entry into the central nervous system from the peripheral organs. While prion strains have identical amino acid sequences, strains differ in the incubation period to terminal disease, lesion profile, and biophysical properties, including conformational stability, depending on the strain. In this work, we found that prion strains that neuroinvade were more soluble than the non-neuroinvasive strains, and that sonicating the non-neuroinvasive strains increased the solubility as well as the neuroinvasive properties. Second, we studied cellular pathways reported to be involved in prion spread through the CNS. We found that the ESCRT (Endosomal Complexes Required for Transport) pathway contributes to prion conversion in multivesicular bodies (MVBs) in neurons and prion spread via exosomes. Surprisingly, depletion of ESCRT-0 in the neurons of prion-infected mice worsened disease by accelerating the degeneration of synapses, without detectably increasing conversion of PrPSc. In contrast, depletion of ESCRT-0 in astrocytes or microglia had no effect on PrPSc levels or survival time. Third, we investigated pathways that contribute to prion spread by analyzing the brains of mice in a longitudinal study that included genetic, histological, and biochemical approaches. We found that synaptic alterations in the hippocampus were among the earliest (40% of disease course) and coincided with accumulation of ubiquitinated protein inclusions, suggesting degradative pathways were impaired early in disease. Fourth, we manipulated endolysosomal pathways in prion-infected neuroblastoma cells to further understand the role of both the MVB in prion conversion and exosomes in prion spread. Here, we validated that knocking down Hrs reduced PrPSc, while knocking down Vps35 conversely increased PrPSc. We found that Hrs depletion caused a decrease of PrPSc. Reduction of prion conversion was not due to a decrease in PrPC or an increase in autophagic degradation of PrPSc, or even a loss of spread through exosomes, but instead, a failure of PrPSc internalization into MVBs. This work has uncovered principal features of prion propagation and spread, as well as unveiled the essential role of the ESCRT pathway in both synapse health and neurodegeneration.