A Neuronal Culture System to Detect Prion Synaptotoxicity

<div><p>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 PrP^Sc, 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, PrP^C, by target neurons, and on the presence of a nine-amino acid, polybasic region at the N-terminus of the PrP^C molecule. Both protease-resistant and protease-sensitive forms of PrP^Sc 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 PrP^Sc and testing potential therapeutic agents.</p></div

The N-terminal domain of PrP^C is essential for PrP^Sc-induced dendritic spine loss.

<p>Hippocampal neurons from Tg(Δ23–111) mice (<b>A-D</b>) and Tg(Δ23–31) mice (<b>E-H</b>) (both on the <i>Prn-p</i>^0/0 background) were treated for 24 hr with 4.4 μg/ml of PrP^Sc purified without proteases (<b>B, F</b>), or with an equivalent amount of mock-purified material from uninfected brains (<b>A, E</b>). Neurons were then fixed and stained with Alexa 488-phalloidin. Scale bar in panel F = 20 μm (applicable to panels A, B, E). Pooled measurements of spine number (<b>C, G</b>) and area (<b>D, H</b>) were collected from 20–24 cells from 4 independent experiments. N.S., not significantly different by Student’s t-test.</p

Purified PrP^Sc, prepared without proteases, causes PrP^C-dependent spine loss.

<p>(<b>A</b>) Silver stain and Western blot analysis (using anti-PrP antibody D18) of PrP^Sc purified from scrapie-infected brains without proteases, and mock-purified material from uninfected brains. Lane M, molecular size markers in kDa. Hippocampal neurons from wild-type (WT) mice (<b>B, C</b>) and PrP knockout (<i>Prn-p</i>^0/0) mice (<b>D, E</b>) were treated for 24 hr with 4.4 μg/ml of purified PrP^Sc (<b>C, E</b>), or with an equivalent amount of material mock-purified from uninfected brains (<b>B, D</b>). Neurons were then fixed and stained with Alexa 488-phalloidin. Scale bar in panel E = 20 μm (applicable to panels B-D). Pooled measurements of spine number (<b>F</b>) and area (<b>G</b>) were collected from 22–25 cells from 4 independent experiments. ***p<0.001 by Student’s t-test; N.S., not significantly different.</p

Purified PrP^Sc, prepared using pronase E, causes PrP^C-dependent spine loss.

<p>(<b>A</b>) Silver stain and Western blot analysis (using anti-PrP antibody IPC1) of PrP^Sc purified from scrapie-infected brains using pronase E, and mock-purified material from uninfected brains. Lane M, molecular size markers in kDa. Hippocampal neurons from wild-type (WT) mice (<b>B, C</b>) and PrP knockout (<i>Prn-p</i>^0/0) mice (<b>D, E</b>) were treated for 24 hr with 4.4 μg/ml of purified PrP^Sc (<b>C, E</b>), or with an equivalent amount of material mock-purified from uninfected brains (<b>B, D</b>). Neurons were then fixed and stained with Alexa 488-phalloidin. Scale bar in panel E = 20 μm (applicable to panels B-D). Pooled measurements of spine number (<b>F</b>) and area (<b>G</b>) were collected from 16–18 cells from 3 independent experiments. ***p<0.001 or *p<0.05 by Student’s t-test; N.S., not significantly different.</p

PK-digested PrP^Sc causes dendritic spine loss.

<p>(<b>A</b>) Silver stain and Western blot (using anti-PrP antibody D18) of a PrP^Sc sample and a mock-purified control sample, after digestion with PK. Lane M, molecular size markers in kDa. Hippocampal neurons from wild-type (WT) mice (<b>B, C</b>) and PrP knockout (<i>Prn-p</i>^0/0) mice (<b>D, E</b>) were treated for 24 hr with 4.4 μg/ml of purified, PK-treated PrP^Sc (<b>C, E</b>), or with an equivalent amount of mock-purified sample (<b>B, D</b>). Neurons were then fixed and stained with Alexa 488-phalloidin. Scale bar in panel E = 20 μm (applicable to panels B-D). Pooled measurements of spine number (<b>F</b>) and area (<b>G</b>) were collected from 20–24 cells from 3 independent experiments. ***p<0.001 by Student’s t-test; N.S., not significantly different.</p