The rat striatum responds to nigro-striatal degeneration via the increased expression of proteins associated with growth and regeneration of neuronal circuitry

Abstract

Background: Idiopathic Parkinson's disease is marked by degeneration of dopamine neurons projecting from the substantia nigra to the striatum. Although proteins expressed by the target striatum can positively affect the viability and growth of dopaminergic neurons, very little is known about the molecular response of the striatum as nigro-striatal denervation progresses. Here, iTRAQ labelling and MALDI TOF/TOF mass spectrometry have been used to quantitatively compare the striatal proteome of rats before, during, and after 6-OHDA induced dopamine denervation.

Results: iTRAQ analysis revealed the differential expression of 50 proteins at 3 days, 26 proteins at 7 days, and 34 proteins at 14 days post-lesioning, compared to the unlesioned striatum. While the denervated striatum showed a reduced expression of proteins associated with the loss of dopaminergic input (e.g., TH and DARPP-32), there was an increased expression of proteins associated with regeneration and growth of neurites (e.g., GFAP). In particular, the expression of guanine deaminase (GDA, cypin) - a protein known to be involved in dendritic branching - was significantly increased in the striatum at 3, 7 and 14 days post-lesioning (a finding verified by immunohistochemistry).

Conclusions: Together, these findings provide evidence to suggest that the response of the normal mammalian striatum to nigro-striatal denervation includes the increased expression of proteins that may have the capacity to facilitate repair and growth of neuronal circuitry.

Keywords: DARPP-32; GFAP; Guanine deaminase; Neurofilament; Nigro-striatal degeneration; Parkinson’s disease; Proteomics; Regeneration; Striatum; iTRAQ.

Figure 1

A schematic diagram illustrating medial forebrain bundle lesioning used to detach the striatum from dopaminergic innervation. A sagittal section from a non-lesioned rat brain, stained with an antibody to tyrosine hydroxylase (TH), highlights how dopaminergic cells residing in the substantia nigra pars compacta (SNc) extend long axonal fibers through the medial forebrain bundle on their way to innervating the striatum (Str). For lesioning, a small glass capillary (vertical arrow) was filled with 6-OHDA and lowered to a point between the SNc and Str (i.e., along the medial forebrain bundle). The 6-OHDA was slowly injected (black dot with halo surrounding) to produce a gradual degeneration of SNc dopamine neurons and their axonal innervation of the striatum. Rostral is left, dorsal is up. Scale bar = 2 mm.

Figure 2

Tyrosine hydroxylase (TH) immunoreactivity decreases by 3 days post-lesioning and is undetectable after 7 and 14 days post-lesioning. Tyrosine hydroxylase (TH) immunoreactivity in coronal sections through the striatum of (A) unlesioned), (B) 3 day, (C) 7 day, and (D) 14 day post-lesioned animals, highlights the ongoing loss of dopaminergic input to the striatum at time points used for proteomic analysis. Though there is a noticeable staining of numerous TH + process in the striatum of the unlesioned brain (A), only few swollen, varicose TH + process remain in the striatum at 3 days post 6-OHDA lesioning (arrow in B). As expected, there was no detectable staining of TH in the striatum after 7 days post lesioning (C), and this remained unchanged at 14 days (D) post 6-OHDA injection to the medial forebrain bundle. Scale bars in A-D = 10 μm. Striatal protein extracts were subjected to SDS-PAGE and transferred to nitrocellulose by electroblotting blotting (E). The blot was probed with an antibody against tyrosine hydroxylase – TH, with beta III tubulin as an internal loading control. PL = post 6-OHDA lesioning.

Figure 3

A graph illustrating the functional annotations that were assigned to the filtered lists of differentially expressed proteins using Ingenuity Pathway Analysis software (IPA).

Figure 4

Glial fibrillary acidic protein (GFAP) immunoreactivity is increased at 3, 7 and 14 days post-lesioning. Glial fibrillary acidic protein (GFAP) immunoreactivity in coronal sections through the unlesioned and lesioned side of striatum from control (unlesioned), 3 days-, 7 days-, and 14 days-post-lesioned animals. At 3 days following 6-OHDA injection along the medial forebrain bundle, widespread and intense GFAP immunoreactivity can be seen in astrocytes throughout the entire striatal complex. Though this level of astrocyte reactivity had subsided somewhat by 7 days post lesioning, the level of GFAP expression in the striatum did not return to the levels seen in the unlesioned brain even after 14 days post lesioning. Scale bar for figures = 10 μm.

Figure 5

Western blotting verifies the expression levels of several proteins that were identified by iTRAQ analysis. Striatal protein extracts were subjected to SDS-PAGE and transferred to nitrocellulose either by electroblotting blotting (B), or by diffusion blotting to produce two identical blots from one gel (A, C, and D). Blots were probed with antibodies against (A) glial fibrillary acidic protein - GFAP, (B) neurofilament heavy polypeptide - NFh, (C) growth associated protein 43 - GAP43, or (D) dopamine- or cAMP- regulated neuronal phosphoprotein - DARPP-32, with beta III tubulin as an internal loading control in each case. PL = post 6-OHDA lesioning.

Figure 6

Guanine deaminase (GDA) is increased in the striatum at 7 and 14 days post-lesioning. Guanine deaminase (GDA) immunoreactivity in coronal sections through the striatum of (A, E, and I) unlesioned, (B, F, and J) 3 day, (C, G, and K) 7 day, and (D, H, and L) 14 day post-lesioned animals. High power, confocal images of the (top row) dorsal, (middle row) medial, and (bottom row) ventral portions of the striatum were taken to both see the fine staining by GDA immunoreactive fibers, and document the protein’s expression throughout the striatum. Though fiber bundles (“striations”) of the striatum were highly reactive for GDA in control and lesioned brains (arrows in E), there was only low levels of reactivity within the striatal grey matter of the unlesioned brain. Immunoreactivity for GDA in the brain 3 days post-lesioning(B, F, and J) suggests some change in the protein’s expression in the grey matter, however, an increase in GDA immunoreactivity was not notable until 7 days post 6-OHDA lesioning (C, G, and K). At this stage, and at 14 days post-lesioning (D, H, and L), numerous fibers, with long thick processes (suggestive of dendrites) were stained throughout the dorsal, medial, and ventral grey matter of the entire striatum. Scale bars for each figure are shown at the bottom right of each image.