Ruth G. Perez

Mutagenesis identifies new signals for β-amyloid precursor protein endocytosis, turnover, and the generation of secreted fragments, including Aβ42

It has long been assumed that the C-terminal motif, NPXY, is the internalization signal for β-amyloid precursor protein (APP) and that the NPXY tyrosine (Tyr743 by APP751 numbering, Tyr682 in APP695) is required for APP endocytosis. To evaluate this tenet and to identify the specific amino acids subserving APP endocytosis, we mutated all tyrosines in the APP cytoplasmic domain and amino acids within the sequence GYENPTY (amino acids 737–743). Stable cell lines expressing these mutations were assessed for APP endocytosis, secretion, and turnover. Normal APP endocytosis was observed for cells expressing Y709A, G737A, and Y743A mutations. However, Y738A, N740A, and P741A or the double mutation of Y738A/P741A significantly impaired APP internalization to a level similar to that observed for cells lacking nearly the entire APP cytoplasmic domain (ΔC), arguing that the dominant signal for APP endocytosis is the tetrapeptide YENP. Although not an APP internalization signal, Tyr743 regulates rapid APP turnover because half-life increased by 50% with the Y743A mutation alone. Secretion of the APP-derived proteolytic fragment, Aβ, was tightly correlated with APP internalization, such that Aβ secretion was unchanged for cells having normal APP endocytosis but significantly decreased for endocytosis-deficient cell lines. Remarkably, secretion of the Aβ42 isoform was also reduced in parallel with endocytosis from internalization-deficient cell lines, suggesting an important role for APP endocytosis in the secretion of this highly pathogenic Aβ species.

α-Synuclein activation of protein phosphatase 2A reduces tyrosine hydroxylase phosphorylation in dopaminergic cells

α-Synuclein is an abundant presynaptic protein implicated in neuronal plasticity and neurodegenerative diseases. Although the function of α-synuclein is not thoroughly elucidated, we found that α-synuclein regulates dopamine synthesis by binding to and inhibiting tyrosine hydroxylase, the rate limiting enzyme in dopamine synthesis. Understanding α-synuclein function in dopaminergic cells should add to our knowledge of this key protein, which is implicated in Parkinsons disease and other disorders. Herein, we report a mechanism by which α-synuclein diminishes tyrosine hydroxylase phosphorylation and activity in stably transfected dopaminergic cells. Short-term regulation of tyrosine hydroxylase depends on the phosphorylation of key seryl residues in the amino-terminal regulatory domain of the protein. Of these, Ser40 contributes significantly to tyrosine hydroxylase activation and dopamine synthesis. We observed that α-synuclein overexpression caused reduced Ser40 phosphorylation in MN9D cells and inducible PC12 cells. Ser40 is phosphorylated chiefly by the cyclic AMP-dependent protein kinase PKA and dephosphorylated almost exclusively by the protein phosphatase, PP2A. Therefore, we measured the impact of α-synuclein overexpression on levels and activity of PKA and PP2A in our cells. PKA was unaffected by α-synuclein. PP2A protein levels also were unchanged, however, the activity of PP2A increased in parallel with α-synuclein expression. Inhibition of PP2A dramatically increased Ser40 phosphorylation only in α-synuclein overexpressors in which α-synuclein was also found to co-immunoprecipitate with PP2A. Together the data reveal a functional interaction between α-synuclein and PP2A that leads to PP2A activation and underscores a key role for α-synuclein in protein phosphorylation.

Enhanced Release of Amyloid -Protein from Codon 670/671 Swedish Mutant -Amyloid Precursor Protein Occurs in Both Secretory and Endocytic Pathways

The mutation at codons 670/671 of β-amyloid precursor protein (βPP) dramatically elevates amyloid β-protein (Aβ) production. Since increased Aβ may be responsible for the disease phenotype identified from a Swedish kindred with familial Alzheimers disease, evaluation of the cellular mechanism(s) responsible for the enhanced Aβ release may suggest potential therapies for Alzheimers disease. In this study, we analyzed Chinese hamster ovary cells stably transfected with either wild type βPP (βPP-wt) or “Swedish” mutant βPP (βPP-sw) for potential differences in βPP processing. We confirmed that increased amounts of Aβ and a β-secretase-cleaved COOH-terminally truncated soluble βPP (βPP) were secreted from βPP-sw cells. As shown previously for βPP-wt cells, Aβ was released more slowly than the secretion of βPP from surface-labeled βPP-sw cells, indicating that endocytosis of cell surface βPP is one source of Aβ production. In contrast, by [S]methionine metabolic labeling, the rates of Aβ and βPP release were virtually identical for both cell lines. In addition, the identification of intracellular βPP and Aβ shortly after pulse labeling suggests that Aβ is produced in the secretory pathway. Interestingly, more Aβ was present in medium from βPP-sw cells than βPP-wt cells after either cell surface iodination or [S]methionine labeling, indicating that βPP-sw cells have enhanced Aβ release in both the endocytic and secretory pathways. Furthermore, a variety of drug treatments known to affect protein processing similarly reduced Aβ release from both βPP-wt and βPP-sw cells. Taken together, the data suggest that the processing pathway for βPP is similar for both βPP-wt and βPP-sw cells and that increased Aβ production by βPP-sw cells arises from enhanced cleavage of mutant βPP by β-secretase, the as-yet unidentified enzyme(s) that cleaves at the NH terminus of Aβ.

Could a loss of α-synuclein function put dopaminergic neurons at risk?

The α‐synuclein gene is implicated in Parkinsons disease, the symptoms of which occur after a marked loss of substantia nigra dopamine neurons. While the function of α‐synuclein is not entirely elucidated, one function appears to be as a normal regulatory protein that can bind to and inhibit tyrosine hydroxylase, the rate‐limiting enzyme in dopamine synthesis. Soluble α‐synuclein levels may be diminished in Parkinsons disease substantia nigra dopamine neurons both by reduced expression and by α‐synuclein aggregation as Lewy bodies and Lewy neurites form. The loss of functional α‐synuclein may then result in dysregulation of tyrosine hydroxylase, dopamine transport and dopamine storage, resulting in excess cytosolic dopamine. Because dopamine and its metabolites are reactive molecules capable of generating highly reactive quinones and reactive oxygen species, a failure to package dopamine into vesicles could cause irreversible damage to cellular macromolecules and contribute to resultant neurotoxicity. This review focuses on how a loss of normal α‐synuclein function may contribute to the dopamine‐related loss of substantia nigra neurons during Parkinsons disease pathogenesis.

Increased dopamine turnover after partial loss of dopaminergic neurons: compensation or toxicity?

6-Hydroxydopamine (6-OHDA) has proven a valuable tool in the study of Parkinsons disease (PD); it has also served to emphasize the possibility that this disorder may result in part from the sort of oxidative stress that 6-OHDA exerts on dopamine neurons. In this review we comment on several lines of our research related to the role of oxidative stress in PD, research that has benefited greatly from the work of Gerald Cohen whose memory we honor at this symposium. First, we discuss our use of 6-OHDA to produce an animal model of PD; second, we comment on our studies on dopamines neurotoxic effects; and finally, we discuss our finding that tyrosine hydroxylase is regulated in part by an interaction with alpha-synuclein. We suggest that PD is associated with an increase in dopamine turnover, which may not only reduce the immediate symptoms of the disease but also contribute to its progression.

Effects of GDNF on 6-OHDA-induced death in a dopaminergic cell line: Modulation by inhibitors of PI3 kinase and MEK

Parkinsons disease is a neurodegenerative disorder associated with the selective death of dopaminergic neurons. Glial cell line‐derived neurotrophic factor (GDNF) can protect dopaminergic neurons in several parkinsonian models. We used the dopaminergic cell line MN9D to explore the mechanisms underlying GDNF‐mediated protection against the neurotoxin 6‐hydroxydopamine (6‐OHDA). MN9D cell viability was decreased 24 hr after a 15‐min exposure to 6‐OHDA (50–1,000 μM) as revealed by staining with Hoechst reagent and Trypan blue. The addition of GDNF (10 ng/ml) before, during, and after exposure to 6‐OHDA significantly increased the number of viable cells as assessed by Hoechst staining. In contrast, 6‐OHDA‐induced cell membrane damage was unaffected as measured by Trypan blue exclusion. The PI3K specific inhibitor LY294002 (10–50 μM) blocked GDNF‐mediated protection against nuclear condensation, as did the MAPK kinase (MEK) inhibitor U0126 (5– 20 μM). These studies suggest that GDNF can protect dopaminergic cells against some but not all aspects of 6‐OHDA‐induced toxicity by acting through both PI3K and MAPK signaling pathways.

Serine 129 Phosphorylation Reduces the Ability of α-Synuclein to Regulate Tyrosine Hydroxylase and Protein Phosphatase 2A in Vitro and in Vivo

α-Synuclein (a-Syn), a protein implicated in Parkinson disease, contributes significantly to dopamine metabolism. a-Syn binding inhibits the activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. Phosphorylation of TH stimulates its activity, an effect that is reversed by protein phosphatase 2A (PP2A). In cells, a-Syn overexpression activates PP2A. Here we demonstrate that a-Syn significantly inhibited TH activity in vitro and in vivo and that phosphorylation of a-Syn serine 129 (Ser-129) modulated this effect. In MN9D cells, a-Syn overexpression reduced TH serine 19 phosphorylation (Ser(P)-19). In dopaminergic tissues from mice overexpressing human a-Syn in catecholamine neurons only, TH-Ser-19 and TH-Ser-40 phosphorylation and activity were also reduced, whereas PP2A was more active. Cerebellum, which lacks excess a-Syn, had PP2A activity identical to controls. Conversely, a-Syn knock-out mice had elevated TH-Ser-19 phosphorylation and activity and less active PP2A in dopaminergic tissues. Using an a-Syn Ser-129 dephosphorylation mimic, with serine mutated to alanine, TH was more inhibited, whereas PP2A was more active in vitro and in vivo. Phosphorylation of a-Syn Ser-129 by Polo-like-kinase 2 in vitro reduced the ability of a-Syn to inhibit TH or activate PP2A, identifying a novel regulatory role for Ser-129 on a-Syn. These findings extend our understanding of normal a-Syn biology and have implications for the dopamine dysfunction of Parkinson disease.

α-SYNUCLEIN AGGREGATION ALTERS TYROSINE HYDROXYLASE PHOSPHORYLATION AND IMMUNOREACTIVITY: LESSONS FROM VIRAL TRANSDUCTION OF KNOCKOUT MICE

Tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine synthesis, is frequently used as a marker of dopaminergic neuronal loss in animal models of Parkinsons disease (PD). We have been exploring the normal function of the PD-related protein alpha-synuclein (alpha-Syn) with regard to dopamine synthesis. TH is activated by the phosphorylation of key seryl residues in the TH regulatory domain. Using in vitro models, our laboratory discovered that alpha-Syn inhibits TH by acting to reduce TH phosphorylation, which then reduces dopamine synthesis [X.-M. Peng, R. Tehranian, P. Dietrich, L. Stefanis, R.G. Perez, Alpha-synuclein activation of protein phosphatase 2A reduces tyrosine hydroxylase phosphorylation in dopaminergic cells, J. Cell. Sci. 118 (2005) 3523-3530; R.G. Perez, J.C. Waymire, E. Lin, J.J. Liu, F. Guo, M.J. Zigmond, A role for alpha-synuclein in the regulation of dopamine biosynthesis, J. Neurosci. 22 (2002) 3090-3099]. We recently began exploring the impact of alpha-Syn on TH in vivo, by transducing dopaminergic neurons in alpha-Syn knockout mouse (ASKO) olfactory bulb using wild type human alpha-Syn lentivirus. At 3.5-21 days after viral delivery, alpha-Syn expression was transduced primarily in periglomerular dopaminergic neurons. Cells with modest levels of alpha-Syn consistently co-labeled for Total-TH. However, cells bearing aggregated alpha-Syn, as revealed by proteinase K or Thioflavin-S treatment had significantly reduced Total-TH immunoreactivity, but high phosphoserine-TH labeling. On immunoblots, we noted that Total-TH immunoreactivity was equivalent in all conditions, although tissues with alpha-Syn aggregates again had higher phosphoserine-TH levels. This suggests that aggregated alpha-Syn is no longer able to inhibit TH. Although the reason(s) underlying reduced Total-TH immunoreactivity on tissue sections await(s) confirmation, the dopaminergic phenotype was easily verified using phosphorylation-state-specific TH antibodies. These findings have implications not only for normal alpha-Syn function in TH regulation, but also for measuring cell loss that is associated with synucleinopathy.

Neuroprotective effects of linarin through activation of the PI3K/Akt pathway in amyloid-β-induced neuronal cell death.

Linarin, a natural occurring flavanol glycoside derived from Mentha arvensis and Buddleja davidii is known to have anti-acetylcholinesterase effects. The present study intended to explore the neuroprotective effects of linarin against Aβ(25-35)-induced neurotoxicity with cultured rat pheochromocytoma cells (PC12 cells) and the possible mechanisms involved. For this purpose, PC12 cells were cultured and exposed to 30 μM Aβ(25-35) in the absence or presence of linarin (0.1, 1.0 and 10 μM). In addition, the potential contribution of the PI3K/Akt neuroprotective pathway in linarin-mediated protection against Aβ(25-35)-induced neurotoxicity was also investigated. The results showed that linarin dose-dependently increased cell viability and reduced the number of apoptotic cells as measured by MTT assay, Annexin-V/PI staining, JC-1 staining and caspase-3 activity assay. Linarin could also inhibit acetylcholinesterase activity induced by Aβ(25-35) in PC12 cells. Further study revealed that linarin induced the phosphorylation of Akt dose-dependently. Treatment of PC12 cells with the PI3K inhibitor LY294002 attenuated the protective effects of linarin. Furthermore, linarin also stimulated phosphorylation of glycogen synthase kinase-3β (GSK-3β), a downstream target of PI3K/Akt. Moreover, the expression of the anti-apoptotic protein Bcl-2 was also increased by linarin treatment. These results suggest that linarin prevents Aβ(25-35)-induced neurotoxicity through the activation of PI3K/Akt, which subsequently inhibits GSK-3β and up-regulates Bcl-2. These findings raise the possibility that linarin may be a potent therapeutic compound against Alzheimers disease acting through both acetylcholinesterase inhibition and neuroprotection.

Alpha‐synuclein inhibits aromatic amino acid decarboxylase activity in dopaminergic cells

Alpha‐synuclein is a presynaptic protein strongly implicated in Parkinsons disease (PD). Because dopamine neurons are invariably compromised during pathogenesis in PD, we have been exploring the functions of alpha‐synuclein with particular relevance to dopaminergic neuronal cells. We previously discovered reduced tyrosine hydroxylase (TH) activity and minimal dopamine synthesis in stably‐transfected MN9D cells overexpressing either wild‐type or A53T mutant (alanine to threonine at amino acid 53) alpha‐synuclein. TH, the rate‐limiting enzyme in dopamine synthesis, converts tyrosine to l‐dihydroxyphenylalanine (L‐DOPA), which is then converted to dopamine by the enzyme, aromatic amino acid decarboxylase (AADC). We confirmed an interaction between alpha‐synuclein and AADC in striatum. We then sought to determine whether wild‐type or A53T mutant alpha‐synuclein might have affected AADC activity in dopaminergic cells. Using HPLC with electrochemical detection, we measured dopamine and related catechols after L‐DOPA treatments to bypass the TH step. We discovered that while alpha‐synuclein did not reduce AADC protein levels, it significantly reduced AADC activity and phosphorylation in our cells. These novel findings further support a role for alpha‐synuclein in dopamine homeostasis and may explain, at least in part, the selective vulnerability of dopamine neurons that occurs in PD.