Pre-treatment of striatal slices by DCF decreases HDA uptake (a) and dopamine content (b)

The concentration of DCF is indicated at the bottom of respective columns. The results are expressed in kBq/g [H]DA uptake and pmol/mg protein (dopamine content), respectively. Means ± SE of observations are presented. The number of experiments is indicated below the columns.* < 0.05, ** < 0.01, significance versus the control non-pre-treated slice.<p><b>Copyright information:</b></p><p>Taken from "Modulation of dopaminergic neurotransmission in rat striatum upon and diclofenac treatment"</p><p></p><p>Journal of Neurochemistry 2008;105(2):360-368.</p><p>Published online 01 Apr 2008</p><p>PMCID:PMC2324205.</p><p>© 2007 The Authors Journal compilation © 2007 International Society for Neurochemistry</p

Immunostaining for TH in the striatal brain sections of Sprague–Dawley rats

Staining was performed on 40-μm Vibratome sections of the brain tissue blocks containing the upper part of the striatum with adjacent cortex. Fixation was performed by 4%-formaldehyde, 0.5% glutaraldehyde, and 15% saturated picric acid in 0.1 mol/L phosphate buffer, pH 7.4. Dilution of applied TH antibody (Sigma) was 1 : 1000. Detection of antigen–antibody complexes was performed by DAB as chromogen. Original magnification: 10× S, striatum; C, cortex. (a) Control staining. TH-antibody was omitted from the incubation medium. Brain section from untreated animal. (b) Immunostaining for TH in the brain section of untreated animal. Strong immunoreactivity in the striatum. (c) Striatal brain section of a sham-operated Sprague–Dawley rat. Intensity of immunostaining for TH antibody is comparable with the control staining. (d, e, and f) Different intensity of the TH-staining in the striatal sections after long-term (4 weeks) of DCF treatment (selected samples from the results of TH-immunostaining of eight treated animals). (d) Significantly weaker immunoreactivity in the striatum (two cases). (e) Intensity of the TH-staining is comparable with the staining found in the striatal sections of sham-operated or control animals (two cases). (f) TH-immunoreactivity is stronger after DCF treatment than control (four cases).<p><b>Copyright information:</b></p><p>Taken from "Modulation of dopaminergic neurotransmission in rat striatum upon and diclofenac treatment"</p><p></p><p>Journal of Neurochemistry 2008;105(2):360-368.</p><p>Published online 01 Apr 2008</p><p>PMCID:PMC2324205.</p><p>© 2007 The Authors Journal compilation © 2007 International Society for Neurochemistry</p

Chronic DCF treatment decreases ATP content (a) and the energy charge (b) of striatal slices

Sham controls, open bars; DCF i.v. treated animals, filled bars. The number of experiments is given in parentheses. Means ± SE of observations are presented.* < 0.05, ** < 0.01, significance versus the sham-operated control.<p><b>Copyright information:</b></p><p>Taken from "Modulation of dopaminergic neurotransmission in rat striatum upon and diclofenac treatment"</p><p></p><p>Journal of Neurochemistry 2008;105(2):360-368.</p><p>Published online 01 Apr 2008</p><p>PMCID:PMC2324205.</p><p>© 2007 The Authors Journal compilation © 2007 International Society for Neurochemistry</p

Autophagy inhibition promotes SNCA/alpha-synuclein release and transfer via extracellular vesicles with a hybrid autophagosome-exosome-like phenotype

<p>The autophagy-lysosome pathway (ALP) regulates intracellular homeostasis of the cytosolic protein SNCA/alpha-synuclein and is impaired in synucleinopathies, including Parkinson disease and dementia with Lewy bodies (DLB). Emerging evidence suggests that ALP influences SNCA release, but the underlying cellular mechanisms are not well understood. Several studies identified SNCA in exosome/extracellular vesicle (EV) fractions. EVs are generated in the multivesicular body compartment and either released upon its fusion with the plasma membrane, or cleared via the ALP. We therefore hypothesized that inhibiting ALP clearance 1) enhances SNCA release via EVs by increasing extracellular shuttling of multivesicular body contents, 2) alters EV biochemical profile, and 3) promotes SNCA cell-to-cell transfer. Indeed, ALP inhibition increased the ratio of extra- to intracellular SNCA and upregulated SNCA association with EVs in neuronal cells. Ultrastructural analysis revealed a widespread, fused multivesicular body-autophagosome compartment. Biochemical characterization revealed the presence of autophagosome-related proteins, such as LC3-II and SQSTM1. This distinct “autophagosome-exosome-like” profile was also identified in human cerebrospinal fluid (CSF) EVs. After a single intracortical injection of SNCA-containing EVs derived from CSF into mice, human SNCA colocalized with endosome and neuronal markers. Prominent SNCA immunoreactivity and a higher number of neuronal SNCA inclusions were observed after DLB patient CSF EV injections. In summary, this study provides compelling evidence that a) ALP inhibition increases SNCA in neuronal EVs, b) distinct ALP components are present in EVs, and c) CSF EVs transfer SNCA from cell to cell in vivo. Thus, macroautophagy/autophagy may regulate EV protein composition and consequently progression in synucleinopathies.</p