Persistent changes in neuronal structure and synaptic plasticity caused by proton irradiation

Abstract

Cranial radiotherapy is used routinely to control the growth of primary and secondary brain tumors, but often results in serious and debilitating cognitive dysfunction. In part due to the beneficial dose depth distributions that may spare normal tissue damage, the use of protons to treat CNS and other tumor types is rapidly gaining popularity. Astronauts exposed to lower doses of protons in the space radiation environment are also at risk for developing adverse CNS complications. To explore the consequences of whole body proton irradiation, mice were subjected to 0.1 and 1 Gy and analyzed for morphometric changes in hippocampal neurons 10 and 30 days following exposure. Significant dose-dependent reductions (~33 %) in dendritic complexity were found, when dendritic length, branching and area were analyzed 30 days after exposure. At equivalent doses and times, significant reductions in the number (~30 %) and density (50-75 %) of dendritic spines along hippocampal neurons of the dentate gyrus were also observed. Immature spines (filopodia, long) exhibited the greatest sensitivity (1.5- to 3-fold) to irradiation, while more mature spines (mushroom) were more resistant to changes over a 1-month post-irradiation timeframe. Irradiated granule cell neurons spanning the subfields of the dentate gyrus showed significant and dose-responsive reductions in synaptophysin expression, while the expression of postsynaptic density protein (PSD-95) was increased significantly. These findings corroborate our past work using photon irradiation, and demonstrate for the first time, dose-responsive changes in dendritic complexity, spine density and morphology and synaptic protein levels following exposure to low-dose whole body proton irradiation.

Fig. 1. Reduced dendritic complexity of granule cell layer (GCL) neurons 10 and 30 days after irradiation

(A–C), Examples of deconvoluted eGFP positive GCL neurons showing dendrites orientated vertically and traversing the molecular layer (ML). (D–F), Deconvoluted 3D reconstructed images of A–C respectively, with dendrites containing spines projecting into the ML (sky blue, cell body; green, dendrites; blue, branch points; red, spines). (G) dendritic branching numbers, (H) dendritic branch points, (I) total dendritic length, and (J) total dendritic area quantified at 10 and 30 days after exposure to 0.1 and 1.0 Gy.

Fig. 2. Radiation-induced reductions in dendrite spine density

(A–E), Representative images 3D reconstructed dendritic segments (green) containing spines (red). (F–H), Quantified dendritic spine parameters including spine number (F), spine density (G) and spine volume (H) along GCL neurons 10 or 30 days after irradiation.

Fig. 3. Radiation-induced changes in the number of morphologically distinct spines

Reconstructed dendritic segments showing the radiation-induced reductions in filopodia (white, A–C) along with other spine types after irradiation, using the color-coded classification shown (D). (E–F), Quantified spine types including immature filopodia along with more mature long, mushroom and stubby morphologies 10 (E) and 30 (F) days following irradiation.

Fig. 4. Up-regulation of PSD-95 expression following irradiation

Fluorescence micrographs show that irradiation leads to increased expression of PSD-95 puncta (red) in GCL neurons of the ML at both 10 (C, D) and 30 (E, F) days after irradiation compared to controls (A). (B) Quantified PSD-95 puncta at 10 and 30 days after exposure to 0.1 and 1.0 Gy.

Fig. 5. Down-regulation of the presynaptic marker synaptophysin following irradiation

Representative images of synaptophysin expression show reduced expression in the DH (blue, cell bodies stained with DAPI; red, synaptophysin expression) at both 10 (C, D) and 30 (E, F) days after irradiation compared to controls (A). (B) Quantified synaptophysin staining at 10 and 30 days after exposure to 0.1 and 1.0 Gy.

Fig. 6

Schematic representation of consequences of photon and proton irradiation-induced deterioration of structural and synaptic parameters of hippocampal neurons that eventually perpetuate into cognitive dysfucntion.