Lysosomal pathology associated with ?-synuclein accumulation in transgenic models using an eGFP fusion protein

Abstract

Disorders with Lewy body (LB) formation, such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB), are characterized by alpha-synuclein accumulation in the neuronal cell body. Recent studies have suggested that in addition to LBs, alpha-synuclein might accumulate more widely throughout the neurons and their processes, leading to neurodegeneration and functional impairment. The precise patterns of alpha-synuclein accumulation in vivo, however, and its relationship with subcellular neuronal alterations such as lysosomal pathology are not completely clear. To this end, we developed transgenic (tg) in vivo and in vitro models expressing a stable enhanced green fluorescent protein (eGFP) tagged in the C-terminal site of a human (h)alpha-synuclein construct under the regulatory control of the platelet-derived growth factor-beta (PDGFbeta) promoter and carried out confocal, ultrastructural, and biochemical studies. In tg mice, confocal studies demonstrated a wide distribution of halpha-synuclein-eGFP in the neuronal cell bodies, axons, and presynaptic terminals. In several neuronal cell bodies and their neurites, halpha-synuclein-eGFP was found not only as inclusions but also as discrete granular structures that in double-labeling studies colocalized with antibodies against halpha-synuclein and the lysosomal marker cathepsin D. Consistent with these findings, ultrastructural analysis showed that halpha-synuclein-eGFP overexpression resulted in the accumulation of electrodense inclusions and laminated bodies suggestive of lysosomal pathology, and that the halpha-synuclein-eGFP protein was more abundant in the lysosomal fractions of the tg animals. Taken together, these findings support the notion that enhanced visualization of alpha-synuclein utilizing a hybrid eGFP molecule reveals a more widespread accumulation of this molecule in several neuronal compartments, promoting lysosomal dysfunction. Furthermore, the PDGFbeta-halpha-synuclein-eGFP tg model might be a valuable tool in testing new treatments for LBD in a fast and reliable manner.

Direct Transfer of α-Synuclein from Neuron to Astroglia Causes Inflammatory Responses in Synucleinopathies

“…Comparisons for the patterns of ␣-synuclein distribution were performed in the brains of our model mimicking AD-like pathology by expressing the human mutated amyloid precursor protein (APP tg, line 41) (n ϭ 6, 6 months old) under the thy1 promoter (25). In addition, studies of the re-distribution of ␣-synuclein were performed in the PDGF␣-syn-GFP tg mouse model (line 78) (n ϭ 6, 6 months old) (26), because in these mice the fused GFP protein expected to reduce the neuronal release of ␣-synuclein compared the PGDF␣-syn tg lines (line D). Animal protocols were approved by the University of California San Diego Institutional Animal Use and Care Committee.…”

Neuropathological Alterations in Alzheimer Disease

“…Specifically, Ab promotes the oligomerization and toxic conversion of a-synuclein (Masliah et al 2001b;Mandal et al 2006), Ab exacerbates the deficits associated with a-synuclein accumulation, Ab and a-synuclein colocalize in membrane and caveolar fractions, and Ab stabilizes a-synuclein multimers that might form channel-like structures in the membrane (Tsigelny et al 2007(Tsigelny et al , 2008. Both lysosomal leakage (Nixon and Cataldo 2006) and oxidative stress (Smith et al 1996) appear to be involved in the process of neurotoxicity and pathological interactions between Ab and a-synuclein (Rockenstein et al 2005).…”

Neuroprotective Effects of Regulators of the Glycogen Synthase Kinase-3β Signaling Pathway in a Transgenic Model of Alzheimer's Disease Are Associated with Reduced Amyloid Precursor Protein Phosphorylation

“…To analyze the integrity of the dendritic arbor, briefly as described previously (Rockenstein et al, 2005b), blind-coded 40-m-thick vibratome sections from mouse brains fixed in 4% paraformaldehyde were immunolabeled with the mouse monoclonal antibody against microtubule-associated protein-2 (MAP2) (dendritic marker, 1:40; Chemicon), as described previously . After overnight incubation with the primary antibodies, sections were incubated with fluorescein isothiocyanate (FITC)-conjugated horse anti-mouse IgG secondary antibody (1:75; Vector Laboratories, Burlingame, CA), transferred to SuperFrost slides (Fisher Scientific, Tustin, CA), and mounted under glass coverslips with anti-fading media (Vector Laboratories).…”