α-Synuclein accumulation and pathology in Parkinson's disease typically display a caudo-rostral pattern of progression, involving neuronal nuclei in the medulla oblongata at the earliest stages. In this study, selective expression and accumulation of human α-synuclein within medullary neurons was achieved via retrograde transport of adeno-associated viral vectors unilaterally injected into the vagus nerve in the rat neck. The exogenous protein progressively spread toward more rostral brain regions where it could be detected within axonal projections. Propagation to the pons, midbrain and forebrain followed a stereotypical pattern of topographical distribution. It affected areas such as the coeruleus–subcoeruleus complex, dorsal raphae, hypothalamus and amygdala ipsilateral and, to a lesser extent, contralateral to the injection side. Spreading was accompanied by evidence of neuritic pathology in the form of axonal varicosities intensely immunoreactive for human α-synuclein and containing Thioflavin-S-positive fibrils. Thus, overexpression of human α-synuclein in the lower brainstem is sufficient to induce its long-distance caudo-rostral propagation, recapitulating features of Parkinson's disease and mechanisms of disease progression.

Alzheimer disease amyloid ␤-peptide (A␤) is generated via proteolytic processing of the ␤-amyloid precursor protein by ␤-and ␥-secretase. ␥-Secretase can be blocked by selective inhibitors but can also be modulated by a subset of non-steroidal antiinflammatory drugs, including sulindac sulfide. These drugs selectively reduce the generation of the aggregation-prone 42-amino acid A␤ 42 and concomitantly increase the levels of the rather benign A␤ 38 . Here we show that A␤ 42 and A␤ 38 generation occur independently from each other. The amount of A␤ 42 produced by cells expressing 10 different familial Alzheimer disease (FAD)-associated mutations in presenilin (PS) 1, the catalytic subunit of ␥-secretase, appeared to correlate with the respective age of onset in patients. However, A␤ 38 levels did not show a negative correlation with the age of onset. Modulation of ␥-secretase activity by sulindac sulfide reduced A␤ 42 in the case of wild type PS1 and two FAD-associated PS1 mutations (M146L and A285V). The remaining eight PS1 FAD mutants showed either no reduction of A␤ 42 or only rather subtle effects. Strikingly, even the mutations that showed no effect on A␤ 42 levels allowed a robust increase of A␤ 38 upon treatment with sulindac sulfide. Similar observations were made for fenofibrate, a compound known to increase A␤ 42 and to decrease A␤ 38 . For mutants that predominantly produce A␤ 42 , the ability of fenofibrate to further increase A␤ 42 levels became diminished, whereas A␤ 38 levels were altered to varying extents for all mutants analyzed. Thus, we conclude that A␤ 38 and A␤ 42 production do not depend on each other. Using an independent non-steroidal anti-inflammatory drug derivative, we obtained similar results for PS1 as well as for PS2. These in vitro results were confirmed by in vivo experiments in transgenic mice expressing the PS2 N141I FAD mutant. Our findings therefore have strong implications on the selection of transgenic mouse models used for screening of the A␤ 42 -lowering capacity of ␥-secretase modulators. Furthermore, human patients with certain PS mutations may not respond to ␥-secretase modulators.Alzheimer disease is the most abundant form of dementia, and increasing numbers of patients are to be expected in the near future. Amyloid ␤-peptide (A␤) 5 is a central player in the disease pathology. Originally it was purified as the building block of the disease-defining amyloid plaques. Now it is becoming clear that amyloid plaques are probably not the major neurotoxic entity in the disease rather this is an assembly of soluble oligomeric A␤ species (1). These assemblies initiate the so-called amyloid cascade and finally induce abnormal phosphorylation of tau and subsequent formation of paired helical filaments (2). A␤ is generated by proteolytic processing of the ␤-amyloid precursor protein (APP). Two proteases, ␤-secretase and ␥-secretase, perform the cleavages on the N and C termini of the A␤ domain, respectively (3). ␤-Secretase is a conventional aspartyl protease, whereas ␥-secretase i...

Phospho-Ser129 α-synuclein is the modified form of α-synuclein that occurs most frequently within Parkinson's disease pathological inclusions. Here we demonstrate that the antidiabetic drug metformin significantly reduces levels of phospho-Ser129 α-synuclein and the ratio of phospho-Ser129 α-synuclein to total α-synuclein. This effect was documented in vitro in SH-SY5Y and HeLa cells as well as in primary cultures of hippocampal neurons. In vitro work also elucidated the mechanisms underlying metformin's action. Following metformin exposure, decreased phospho-Ser129 α-synuclein was not strictly dependent on induction of AMP-activated protein kinase, a primary target of the drug. On the other hand, metformin-induced phospho-Ser129 α-synuclein reduction was consistently associated with inhibition of mammalian target of rapamycin (mTOR) and activation of protein phosphatase 2A (PP2A). Evidence supporting a key role of mTOR/PP2A signaling included the finding that, similar to metformin, the canonical mTOR inhibitor rapamycin was capable of lowering the ratio of phospho-Ser129 α-synuclein to total α-synuclein. Furthermore, no decrease in phosphorylated α-synuclein occurred with either metformin or rapamycin when phosphatase activity was inhibited, supporting a direct relationship between mTOR inhibition, PP2A activation and protein dephosphorylation. A final set of experiments confirmed the effectiveness of metformin in vivo in wild-type C57BL/6 mice. Addition of the drug to food or drinking water lowered levels of phospho-Ser129 α-synuclein in the brain of treated animals. These data reveal a new mechanism leading to α-synuclein dephosphorylation that could be targeted for therapeutic intervention by drugs like metformin and rapamycin.

J. Neurochem. (2009) 112, 940–950. Abstract γ‐Secretase is a pivotal intramembrane‐cleaving protease complex and important drug target for Alzheimer’s disease. The protease not only releases small peptides, such as the amyloid‐β peptide, which drives Alzheimer’s disease pathogenesis, but also intracellular domains, which can have critical functions in nuclear signaling. Unlike typical aspartyl proteases, γ‐secretase contains a non‐classical GxGD active site motif in its catalytic subunit presenilin (PS) 1 or PS2. It is not known whether both glycines are of similar functional relevance and why the glycine residues are invariant elements of the motif. Here we identify the N‐terminal glycine of the GxGD motif in PS1, G382, as a critical residue of the active site domain of γ‐secretase. Substitution of G382 by a number of different amino acids abrogated γ‐secretase activity. Only the smallest possible G382A substitution allowed substantial γ‐secretase activity. Depending on the substrate, however, the presence of G382 could become even an absolute functional requirement of γ‐secretase. Very similar results were obtained for the C‐terminal glycine residue (G384) of the GxGD motif. Our data thus identify a requirement for small side chain residues in the active site domain of γ‐secretase and suggest that the glycines of the GxGD motif could be evolutionary conserved to allow cleavage of all possible γ‐secretase substrates, including those, which are highly sensitive to minimal alteration of the PS active site domain. These findings broaden our understanding of γ‐secretase substrate recognition and cleavage, which may prove crucial for therapeutic targeting of the enzyme.

c-Secretase plays a central role in the generation of the Alzheimer disease-causing amyloid b-peptide (Ab) from the b-amyloid precursor protein (APP) and is thus a major Alzheimer′s disease drug target. As several other c-secretase substrates including Notch1 and CD44 have crucial signaling functions, an understanding of the mechanism of substrate recognition and cleavage is key for the development of APP selective c-secretase-targeting drugs. The c-secretase active site domain in its catalytic subunit presenilin (PS) 1 has been implicated in substrate recognition/docking and cleavage. Highly critical in this process is its GxGD active site motif, whose invariant glycine residues cannot be replaced without causing severe functional losses in substrate selection and/or cleavage efficiency. Here, we have investigated the contribution of the less well characterized residue x of the motif (L383 in PS1) to this function. Extensive mutational analysis showed that processing of APP was overall well-tolerated over a wide range of hydrophobic and hydrophilic mutations. Interestingly, however, most L383 mutants gave rise to reduced levels of Ab 37-39 species, and several increased the pathogenic Ab 42/43 species. Several of the Ab 42/43 -increasing mutants severely impaired the cleavages of Notch1 and CD44 substrates, which were not affected by any other L383 mutation. Our data thus establish an important, but compared with the glycine residues of the motif, overall less critical functional role for L383. We suggest that L383 and the flanking glycine residues form a spatial arrangement in PS1 that is critical for docking and/or cleavage of different c-secretase substrates.