BACE1 elevation is associated with aberrant limbic axonal sprouting in epileptic CD1 mice

Abstract

The brain is capable of remarkable synaptic reorganization following stress and injury, often using the same molecular machinery that governs neurodevelopment. This form of plasticity is crucial for restoring and maintaining network function. However, neurodegeneration and subsequent reorganization can also play a role in disease pathogenesis, as is seen in temporal lobe epilepsy and Alzheimer's disease. β-Secretase-1 (BACE1) is a protease known for cleaving β-amyloid precursor protein into β-amyloid (Aβ), a major constituent in amyloid plaques. Emerging evidence suggests that BACE1 is also involved with synaptic plasticity and nerve regeneration. Here we examined whether BACE1 immunoreactivity (IR) was altered in pilocarpine-induced epileptic CD1 mice in a manner consistent with the synaptic reorganization seen during epileptogenesis. BACE1-IR increased in the CA3 mossy fiber field and dentate inner molecular layer in pilocarpine-induced epileptic mice, relative to controls (saline-treated mice and mice 24-48 h after pilocarpine-status), and paralleled aberrant expression of neuropeptide Y. Regionally increased BACE1-IR also occurred in neuropil in hippocampal area CA1 and in subregions of the amygdala and temporal cortex in epileptic mice, colocalizing with increased IR for growth associated protein 43 (GAP43) and polysialylated-neural cell adhesion molecule (PSA-NCAM), but reduced IR for microtubule-associated protein 2 (MAP2). These findings suggest that BACE1 is involved in aberrant limbic axonal sprouting in a model of temporal lobe epilepsy, warranting further investigation into the role of BACE1 in physiological vs. pathological neuronal plasticity.

Fig. 1

Immunolabeling for neuropeptide Y (NPY) and β-secretase-1 (BACE1) increased in the hippocampal formation of epileptic mice. Low and high magnifications mid-hippocampal sections are from a control animal (Saline control) and pilocarpine-treated mice that developed status epilepticus and were sacrificed after surviving 2 days (2 d, post-status) or 2 months (Epileptic, 2 mo). Cell loss in areas CA3 and CA1 was seen in epileptic mice that survived 2 mo (C) as compared to control (A) or 2 d survival (B). Compared to control (D, G), NPY neoexpression in the mossy fiber terminals emerges at 2 d (E, H) but intensified with longer survival time (F, I). Mossy fiber sprouting into the inner molecular layer (indicated by triangles in “I”) was seen at the 2 mo survival time. NPY-labeled interneurons were seen in the cortex and hippocampal formation, including in the hilus (G). In control mice, BACE1 labeling in the control animal was prominent in the region of the mossy fiber pathway (J, M). The reactivity appeared to increase in the mossy fiber terminals from 2 d to 2 mo in the epileptic mice. Aberrant mossy fiber sprouting into the dentate inner molecular layer (indicated by triangles in “O”) was evident at the 2 mo survival point. Increased non-cellular labeling of BACE1 was also present in CA1 in 2 mo epileptic animals (L), with many darkly-labeled swollen terminals and processes present in the vicinity of the pyramidal cell layer (R, asterisks). DG: dentate gyrus; Hi:hilus; GCL: granule cell layer; s.p.: stratum pyramidale. Scale bar (in A)=1 mm for A–F and JL; bar=100 μm for G–I and M–R.

Fig. 2

BACE1 immunolabeling in temporal lobe structures increased after one month of status epilepticus. Compared to control (A–D), neuropil-like BACE1 labeling in epileptic mice appeared regionally in the amygdala (Amy), piriform (pir) and entorhinal cortices (E–G), as indicated by the green circles. Mossy fiber sprouting (MFs) in CA3 and the dentate inner molecular layer was also observed in epileptic mice (H). In addition, a band-like labeling extended from CA3 into CA1 (indicated by large arrows in E–H) from stratum oriens (s.o.), through stratum radiatum (s.r.), but not including the pyramidal cell layer (s.p.) or stratum lucunosum-moleculare (s.l.m.) (I). Darkly labeled swollen puncta and processes were scattered in the hippocampus and piriform cortex (I, K). Arabic numbers indicate cortical layers (J, K). Scale bar (in A)=1 mm in A–C, E–G; bar=250 μm D, H and 100 μm for I–K.

Fig. 3

Double immunofluorescence showed a parallel increase of BACE1 and GAP43 labeling in CA1 and CA3 after one month of status epilepticus. Images A–C, G and H show normal distribution patterns of the two markers in the hippocampal formation, with framed areas enlarged/merged as other panels. In epileptic mice, increased GAP43 and BACE1 immunofluorescence co-occurred in CA3 and extended into the medial part of CA1 (D–F). Note overlap of GAP43 and BACE1 in CA1 shown by circled regions in J and K as well as in the merged image (L). Correlative densitometry revealed significantly increased specific optical density (o.d.) for both GAP43 and BACE1 in stratum oriens/stratum radiatum (s.o.–s.r.) in CA1 and the inner molecular layer [ML(i)] in the epileptics relative to controls (I). Specific o.d. of BACE1, but not GAP43, was reduced in the stratum lucunosum-moleculare (s.l.m.) (I). Bisbenzimide (Bis, blue) nuclear counterstain is included in merged images in C and F. White arrows in K highlight BACE1-positive mossy fiber sprouting. Other abbreviations — GCL: granule cell layer; s.p.=stratum pyramidale; ML(o): outer molecular layer. Scale bar (in A)=500 μm for A, B, D, E; bar=250 μm for the remaining images.

Fig. 4

Double immunofluorescence showed a parallel increase of BACE1 and PSA-NCAM labeling in CA1 and CA3 after one month of status epilepticus. Images A and G are stained with bisbenzimide for orientation. Intense BACE1 and PSA-NCAM labeling is normally present in the mossy fiber terminal field (B–F). In the epileptic mice, increased labeling for both markers occurred in CA3 mossy fiber terminals (note particularly merged image in F) but also extended into CA1 (H–L). Bar graphs depict increased specific optical density (o.d.) of PSA-NCAM (M) and BACE1 (N) immunofluorescence in CA1 and CA3 in epileptic vs. control mice. No differences between groups for either marker were seen in parietal cortex (O). Scale bar (in A)=500 μm for A–C and G–I; bar=250 μm for the remaining images.

Fig. 5

Opposing changes in regional BACE1 and MAP2 labeling in temporal lobe areas after one month of status epilepticus. Panels A and B show the normal pattern of BACE1 and MAP2 immunofluorescence in CA1 and the dentate gyrus from a control mouse. In the epileptic animals, BACE1 labeling increased but MAP1 labeling was reduced in area CA1 (D, E, outlined areas; merged in F) and the piriform/entorhinal cortex (G, H, outlined areas). Densitometry (C) confirmed increased BACE1 and reduced MAP labeling in CA1 in stratum oriens (s.o.), stratum radiatum (s.r.), dentate inner molecular layer [ML(i)], mossy fiber field (MF) in CA3 and the amygdala/piriform cortex in the epileptic mice. Of note, BACE1 and MAP2 densities were both reduced in the stratum lucunosum-moleculare (s.l.m.). Specific optical densities from the epileptic tissues are normalized to the means (i.e., defined as 100%, blue broken line in C) of control samples for corresponding measuring sites. PF: piriform fissure; Ent: entorhinal cortex; Pir: piriform cortex; Amyg: amygdala. Scale bar (in A)= 250 μm, and applies to all image panels.