A Neuronal Culture System to Detect Prion Synaptotoxicity

Abstract

Synaptic pathology is an early feature of prion as well as other neurodegenerative diseases. Although the self-templating process by which prions propagate is well established, the mechanisms by which prions cause synaptotoxicity are poorly understood, due largely to the absence of experimentally tractable cell culture models. Here, we report that exposure of cultured hippocampal neurons to PrPSc, the infectious isoform of the prion protein, results in rapid retraction of dendritic spines. This effect is entirely dependent on expression of the cellular prion protein, PrPC, by target neurons, and on the presence of a nine-amino acid, polybasic region at the N-terminus of the PrPC molecule. Both protease-resistant and protease-sensitive forms of PrPSc cause dendritic loss. This system provides new insights into the mechanisms responsible for prion neurotoxicity, and it provides a platform for characterizing different pathogenic forms of PrPSc and testing potential therapeutic agents.

Identification of Anti-prion Compounds using a Novel Cellular Assay

“…Sc causes a rapid, PrP C -dependent retraction of dendritic spines in cultured hippocampal neurons, a phenomenon that we contend represents an early manifestation of the synaptotoxic effect of prion infection (38). As reported previously, incubation of hippocampal neurons with purified PrP Sc , but not with mock-purified material from uninfected brain, caused a dramatic retraction of dendritic spines, as monitored by staining with Alexa-phalloidin, which stains F-actin in the spines (Fig.…”

“…The fact that these compounds were discovered using the DBCA, which we think is related in some way to alterations in a physiological activity of PrP C , makes it more likely that the compounds will affect processes beyond PrP Sc metabolism. The combination of the DBCA, the ScN2a assay, and the PrP Sc dendritic toxicity assay (38) should allow us to discover additional therapeutic leads that act via novel mechanisms.…”

“…Sc from infected brains were prepared as described previously (38). Neurons were preincubated for 2 h with 5 M JZ107 or JZ103 (or DMSO vehicle for the controls), after which 4.4 g/ml of PrP Sc (or a mock-purified control sample from non-infected brains) was added for 24 h. Neurons were then fixed in 4% paraformaldehyde and stained with Alexa 488-phalloidin (Thermo Fisher) to visualize actin in dendritic spines.…”

DLP1‐dependent mitochondrial fragmentation and redistribution mediate prion‐associated mitochondrial dysfunction and neuronal death

“…Abundant ATP is necessary for cell metabolism and multiple synaptic functions at nerve terminals such as neurite outgrowth and synaptic plasticity (Fang et al ., 2016; Manczak et al ., 2016). After observing that the balance of mitochondrial dynamics is definitely tipped in vitro and in vivo models of prion diseases, we further showed MMP collapse accompanied by ATP loss in PrP 106–126 ‐treated neuron cells in vitro and the hamster prion model in vivo .…”

“…In prion diseases, apart from neuronal loss, earliest and potentially most vital neuropathology changes occur at the level of synapses including synapse loss, morphological changes, and functional abnormality (Mallucci, 2009). Two‐photon imaging studies of living, prion‐infected animals found that retraction of dendritic spines occur early during disease progression (Fuhrmann et al ., 2007; Fang et al ., 2016). Changes in their morphology are now believed to underlie synaptic plasticity associated with learning and memory, as well as degenerative events that occur during aging and neurological diseases (Sala & Segal, 2014; Herms & Dorostkar, 2016).…”