Intracellular fibril formation, calcification, and enrichment of chaperones, cytoskeletal, and intermediate filament proteins in the adult hippocampus CA1 following neonatal exposure to the nonprotein amino acid BMAA

Abstract

The environmental neurotoxin β-N-methylamino-L-alanine (BMAA) has been implicated in the etiology of neurodegenerative disease, and recent studies indicate that BMAA can be misincorporated into proteins. BMAA is a developmental neurotoxicant that can induce long-term learning and memory deficits, as well as regionally restricted neuronal degeneration and mineralization in the hippocampal CA1. The aim of the study was to characterize long-term changes (2 weeks to 6 months) further in the brain of adult rats treated neonatally (postnatal days 9-10) with BMAA (460 mg/kg) using immunohistochemistry (IHC), transmission electron microscopy, and laser capture microdissection followed by LC-MS/MS for proteomic analysis. The histological examination demonstrated progressive neurodegenerative changes, astrogliosis, microglial activation, and calcification in the hippocampal CA1 3-6 months after exposure. The IHC showed an increased staining for α-synuclein and ubiquitin in the area. The ultrastructural examination revealed intracellular deposition of abundant bundles of closely packed parallel fibrils in neurons, axons, and astrocytes of the CA1. Proteomic analysis of the affected site demonstrated an enrichment of chaperones (e.g., clusterin, GRP-78), cytoskeletal and intermediate filament proteins, and proteins involved in the antioxidant defense system. Several of the most enriched proteins (plectin, glial fibrillar acidic protein, vimentin, Hsp 27, and ubiquitin) are known to form complex astrocytic inclusions, so-called Rosenthal fibers, in the neurodegenerative disorder Alexander disease. In addition, TDP-43 and the negative regulator of autophagy, GLIPR-2, were exclusively detected. The present study demonstrates that neonatal exposure to BMAA may offer a novel model for the study of hippocampal fibril formation in vivo.

Fig. 1

Hippocampal CA1 areas of a 6-month rat treated neonatally with BMAA on PND 9–10, showing deposition of birefringent material within neurons (a) were collected using laser capture dissection (b). Examples of collected sample areas are shown in red and control tissue in green (c). The CA1 sample (d) and control tissue (e) were catapulted into the cap of the tube and directly treated with trypsin for a two-step digestion in the capture tubes, and LC-MS/MS was performed

Fig. 2

Histopathological changes in the hippocampal CA1 of rats treated neonatally with BMAA on PND 9–10, as examined at the 3-month survival time point. Degenerating/necrotic neurons contain a basophilic, birefringent material (a) and demonstrated to contain calcium using Alizarin red staining (b). Hypertrophic astrocytes within the same area display positive staining for ubiquitin (c) and GFAP (d). Magnification ×20

Fig. 3

Histopathological changes in the hippocampal CA1 of rats treated neonatally with BMAA on PND 9–10, as examined at the 6-month survival time point. Necrotic neurons are present as basophilic (mineralized) remnants, accompanied by marked astrocytosis (a). Some of the astrocytes display intensely eosinophilic cytoplasm, suggestive of Rosenthal fibers (b). The astrocytes show positive staining for GFAP (c) and ubiquitin (d). Magnifications: ×10 (a), ×20 (c), ×40 (b, d)

Fig. 4

Ultrastructural features of neurons and astrocytes in the hippocampal CA1 of rats treated neonatally with BMAA on PND 9–10, as examined at the 3-month survival time point. A normal control neuron is shown for comparison (a). Bundles of parallel-oriented fibrils are present in the cytoplasm of neurons without (b–d) and with (e) intracellular crystals. The appearance of the crystals is consistent with calcium. The fibrils are present also in axons (f). In addition, an abundance of fibrils is present in the cytoplasm of astrocytes (g–h). Magnifications: ×5,800 (g) ×7,900 (a, b, f), ×25,000 (c, h), ×46,000 (e), ×92,000 (d, f; inset)