Common anti-apoptotic roles of parkin and alpha-synuclein in human dopaminergic cells

Parkin, a product of the gene responsible for autosomal recessive juvenile parkinsonism (AR-JP), is an important player in the pathogenic process of Parkinson's disease (PD). Despite numerous studies including search for the substrate of parkin as an E3 ubiquitin-protein ligase, the mechanism by which loss-of-function of parkin induces selective dopaminergic neuronal death remains unclear. Related to this issue, here we show that antisense knockdown of parkin causes apoptotic cell death of human dopaminergic SH-SY5Y cells associated with caspase activation and accompanied by accumulation of oxidative dopamine (DA) metabolites due to auto-oxidation of DOPA and DA. Forced expression of alpha-synuclein (alpha-SN), another familial PD gene product, prevented accumulation of oxidative DOPA/DA metabolites and cell death caused by parkin loss. Our findings indicate that both parkin and alpha-SN share a common pathway in DA metabolism whose abnormality leads to accumulation of oxidative DA metabolites and subsequent cell death.     

Interaction between tau and alpha-synuclein proteins is impaired in the presence of P301L tau mutation

Deposits of tau and alpha-synuclein are hallmarks of distinct neurodegenerative diseases: tauopathies and alpha-synucleinopathies. Affinity chromatography experiments demonstrated a direct binding of the two proteins, and alpha-synuclein was shown to induce fibrillization of tau. Here, we verify the presence of this physical interaction by using different cellular systems. This binding was abolished by the most common tau mutation (P301L) associated with frontotemporal dementia. We restored the impaired interaction by inducing heat shock proteins 70 and 90. In addition, we show that P301L tau mutation strongly affects tau and alpha-synuclein neuronal distribution.     

Interaction of alpha-synuclein and synphilin-1: effect of Parkinson's disease-associated mutations

alpha-Synuclein is a major component of Lewy bodies, a neuropathological feature of Parkinson's disease. Two alpha-synuclein mutations, Ala53Thr and Ala30Pro, are associated with early onset, familial forms of the disease. Recently, synphilin-1, a protein found to interact with alpha-synuclein by yeast two hybrid techniques, was detected in Lewy bodies. In this study we report the interaction of alpha-synuclein and synphilin-1 in human neuroglioma cells using a sensitive fluorescence resonance energy transfer technique. We demonstrate that the C-terminus of alpha-synuclein is closely associated with the C-terminus of synphilin-1. A weak interaction occurs between the N-terminus of alpha-synuclein and synphilin-1. The familial Parkinson's disease associated mutations of alpha-synuclein (Ala53Thr and Ala30Pro) also demonstrate a strong interaction between their C-terminal regions and synphilin-1. However, compared with wild-type alpha-synuclein, significantly less energy transfer occurs between the C-terminus of Ala53Thr alpha-synuclein and synphilin-1, suggesting that the Ala53Thr mutation alters the conformation of alpha-synuclein in relation to synphilin-1.     

Agrin binds alpha-synuclein and modulates alpha-synuclein fibrillation

Recent studies have begun to investigate the role of agrin in brain and suggest that agrin's function likely extends beyond that of a synaptogenic protein. Particularly, it has been shown that agrin is associated with the pathological lesions of Alzheimer's disease (AD) and may contribute to the formation of beta-amyloid (Abeta) plaques in AD. We have extended the analysis of agrin's function in neurodegenerative diseases to investigate its role in Parkinson's disease (PD). Alpha-synuclein is a critical molecular determinant in familial and sporadic PD, with the formation of alpha-synuclein fibrils being enhanced by sulfated macromolecules. In the studies reported here, we show that agrin binds to alpha-synuclein in a heparan sulfate-dependent (HS-dependent) manner, induces conformational changes in this protein characterized by beta-sheet structure, and enhances insolubility of alpha-synuclein. We also show that agrin accelerates the formation of protofibrils by alpha-synuclein and decreases the half-time of fibril formation. The association of agrin with PD lesions was also explored in PD human brain, and these studies shown that agrin colocalizes with alpha-synuclein in neuronal Lewy bodies in the substantia nigra of PD brain. These studies indicate that agrin is capable of accelerating the formation of insoluble protein fibrils in a second common neurodegenerative disease. These findings may indicate shared molecular mechanisms leading to the pathophysiology in these two neurodegenerative disorders.     

Similar patterns of mitochondrial vulnerability and rescue induced by genetic modification of alpha-synuclein, parkin, and DJ-1 in Caenorhabditis elegans

How genetic and environmental factors interact in Parkinson disease is poorly understood. We have now compared the patterns of vulnerability and rescue of Caenorhabditis elegans with genetic modifications of three different genetic factors implicated in Parkinson disease (PD). We observed that expressing alpha-synuclein, deleting parkin (K08E3.7), or knocking down DJ-1 (B0432.2) or parkin produces similar patterns of pharmacological vulnerability and rescue. C. elegans lines with these genetic changes were more vulnerable than nontransgenic nematodes to mitochondrial complex I inhibitors, including rotenone, fenperoximate, pyridaben, or stigmatellin. In contrast, the genetic manipulations did not increase sensitivity to paraquat, sodium azide, divalent metal ions (Fe(II) or Cu(II)), or etoposide compared with the nontransgenic nematodes. Each of the PD-related lines was also partially rescued by the antioxidant probucol, the mitochondrial complex II activator, D-beta-hydroxybutyrate, or the anti-apoptotic bile acid tauroursodeoxycholic acid. Complete protection in all lines was achieved by combining d-beta-hydroxybutyrate with tauroursodeoxycholic acid but not with probucol. These results show that diverse PD-related genetic modifications disrupt the mitochondrial function in C. elegans, and they raise the possibility that mitochondrial disruption is a pathway shared in common by many types of familial PD.     

A novel mechanism of interaction between alpha-synuclein and biological membranes

Conformational abnormalities and aggregation of alpha-synuclein (alpha-syn) have been linked to the pathogenesis of Parkinson's (PD) and related diseases. It has been shown that alpha-syn can stably bind artificial phospholipid vesicles through alpha-helix formation in its N-terminal repeat region. However, little is known about the membrane interaction in cells. In the current study, we determined the membrane-binding properties of alpha-syn to biological membranes by using bi-functional chemical crosslinkers, which allow the detection of transient, but specific, interactions. By utilizing various point mutations and deletions within alpha-syn, we demonstrated that the membrane interaction of alpha-syn in cells is also mediated by alpha-helix formation in the N-terminal repeat region. Moreover, the PD-linked A30P mutation causes reduced membrane binding, which is concordant with the artificial membrane studies. However, contrary to the interaction with artificial membranes, the interaction with biological membranes is rapidly reversible and is not driven by electrostatic attraction. Furthermore, the interaction of alpha-syn with cellular membranes occurs only in the presence of non-protein and non-lipid cytosolic components, which distinguishes it from the spontaneity of the interaction with artificial membranes. More interestingly, addition of the cytosolic preparation to artificial membranes resulted in the transient, charge-independent binding of alpha-syn similar to the interaction with biological membranes. These results suggest that in cells, alpha-syn is engaged in a fundamentally different mode of membrane interaction than the charge-dependent artificial membrane binding, and the mode of interaction is determined by the intrinsic properties of alpha-syn itself and by the cytoplasmic context.     

Cloning of rat parkin cDNA and distribution of parkin in rat brain

The rat parkin cDNA sequence was characterized after screening a rat hypothalamus cDNA library with a 32P-labeled probe containing the entire open reading frame of the human parkin cDNA. This sequence encompasses 1,576 bp and contains a single open reading frame that encodes a 465-amino acid protein. The rat parkin amino acid sequence exhibits a very striking homology to the human and mouse parkin, with 85 and 95% identity, respectively. Both the N-terminal ubiquitin and the ring-IBR (in between ring)-ring finger domains appear to be highly conserved among rat, human, and mouse parkin. An affinity-purified polyclonal antibody (ASP5p) was generated with a synthetic peptide corresponding to amino acids 295-311 of the parkin sequence, which is identical in the three species. Western blotting revealed that ASP5p recognizes a single 52-kDa band, which corresponds to the molecular mass of the parkin protein. Immunostaining with ASP5p showed that parkin is principally located in the cytoplasm of neurons that are widely distributed in the rat brain. Parkin-immunoreactive neurons abound in structures that are specifically targeted in Parkinson's disease, e.g., subtantia nigra, but are also present in unaffected structures, e.g., cerebellum. Furthermore, parkin-enriched glial cells can be detected in various nuclei of the rat brain. Thus, the role of parkin may be much more global than previously thought on the basis of genetic findings gathered in cases of early-onset parkinsonism.     

Ubiquitin, proteasome and parkin

The ubiquitin-proteasome system (UPS) is important for intracellular proteolysis, and is responsible for a diverse array of biologically important cellular processes, such as cell-cycle progression, signaling cascades and developmental programs. This system is also involved in the protein quality control, which maintains the health of the cell. Thus, the UPS provides a clue for understanding of the molecular mechanisms underlying various neurodegenerative diseases. In the last decade, we witnessed a tremendous progress in uncovering the mechanisms of Parkinson's disease (PD). Of the several genes that can cause familial PD, parkin, the causative gene of autosomal recessive juvenile parkinsonism (ARJP), is of a special interest because it encodes an ubiquitin-protein ligase, which covalently attaches ubiquitin to target proteins, designating them for destruction by the proteasome. This review summarizes recent studies on the UPS pathway with a special reference to parkin, focusing on how parkin is linked to the pathogenesis of ARJP.     

Interaction of the molecular chaperone alphaB-crystallin with alpha-synuclein: effects on amyloid fibril formation and chaperone activity

alpha-Synuclein is a pre-synaptic protein, the function of which is not completely understood, but its pathological form is involved in neurodegenerative diseases. In vitro, alpha-synuclein spontaneously forms amyloid fibrils. Here, we report that alphaB-crystallin, a molecular chaperone found in Lewy bodies that are characteristic of Parkinson's disease (PD), is a potent in vitro inhibitor of alpha-synuclein fibrillization, both of wild-type and the two mutant forms (A30P and A53T) that cause familial, early onset PD. In doing so, large irregular aggregates of alpha-synuclein and alphaB-crystallin are formed implying that alphaB-crystallin redirects alpha-synuclein from a fibril-formation pathway towards an amorphous aggregation pathway, thus reducing the amount of physiologically stable amyloid deposits in favor of easily degradable amorphous aggregates. alpha-Synuclein acts as a molecular chaperone to prevent the stress-induced, amorphous aggregation of target proteins. Compared to wild-type alpha-synuclein, both mutant forms have decreased chaperone activity in vitro against the aggregation of reduced insulin at 37 degrees C and the thermally induced aggregation of betaL-crystallin at 60 degrees C. Wild-type alpha-synuclein abrogates the chaperone activity of alphaB-crystallin to prevent the precipitation of reduced insulin. Interaction between these two chaperones and formation of a complex are also indicated by NMR spectroscopy, size-exclusion chromatography and mass spectrometry. In summary, alpha-synuclein and alphaB-crystallin interact readily with each other and affect each other's properties, in particular alpha-synuclein fibril formation and alphaB-crystallin chaperone action.     

Interaction between Doxycycline and Barbiturates

In a cross-over study of five hospitalized patients the half life of doxycycline was significantly shortened after 10 days'' treatment with phenobarbitone. In five patients on continuous barbiturate therapy the half life of doxycycline was even shorter. Barbiturates or other agents inducing drug metabolism should be used cautiously in combination with doxycycline, since this might result in therapeutically inadequate serum concentrations of the antibiotic.     

Interaction between batrachotoxin and yohimbine

The neurotoxins, batrachotoxin and veratridine, are specific activators of sodium channels and cause an increase in the rate of 22Na uptake in neuroblastoma cells. Yohimbine, an indolakylamine alkaloid, inhibits this batrachotoxin-induced 22Na uptake. The dose-response curve of yohimbine suggest that the inhibitor acts reversibly on a single class of binding sites with dissociation constant of 3--4 x 10(-5) M. The dissociation constant is not affected by depolarization from--41 to 0 mV. Kinetic and equilibrium experiments indicate that yohimbine is a competitive inhibitor of the action of batrachotoxin. These results support the conclusion that yohimbine inhibitis the sodium flux by acting on the channel gating mechanism rather than by occluding the channels.     

Interaction between flavins and alcohols

The effect of alcohols on the spectral properties of riboflavin derivatives in non-polar solvent was studied by various spectroscopic methods in order to support the view point that alcohol may directly interact with the isoalloxazine moiety of FAD and enhance the catalytic activity of D-amino acid oxidase (DAAO). The most likely association complex between alcohol and riboflavin is 1 : 1 stoichiometric complex through the 3-N imino and the 2-C carbonyl groups of the isoalloxazine ring and the hydroxyl group of alcohols. It appears that methanol has a larger association constant than any other alcohols, and the association constant decreases with the increase in carbon number and with the steric requirement of the alkyl group of alcohols.     

Interaction between Levodopa and Methyldopa

The interaction between methyldopa and levodopa was studied in 18 patients with Parkinsonism. Together they produced a fall in blood pressure in doses which when given alone had no effect or only a slight hypotensive effect. Severe hypotension never occurred. It is reasonable to give methyldopa to hypertensive patients on levodopa but the regimen should be initiated in hospital.     

Dopamine covalently modifies and functionally inactivates parkin

Inherited mutations in PARK2, the gene encoding parkin, cause selective degeneration of catecholaminergic neurons in the substantia nigra and locus coeruleus of the brainstem, resulting in early-onset parkinsonism. But the role of parkin in common, sporadic forms of Parkinson disease remains unclear. Here we report that the neurotransmitter dopamine covalently modifies parkin in living dopaminergic cells, a process that increases parkin insolubility and inactivates its E3 ubiquitin ligase function. In the brains of individuals with sporadic Parkinson disease, we observed decreases in parkin solubility consistent with its functional inactivation. Using a new biochemical method, we detected catechol-modified parkin in the substantia nigra but not other regions of normal human brain. These findings show a vulnerability of parkin to modification by dopamine, the principal transmitter lost in Parkinson disease, suggesting a mechanism for the progressive loss of parkin function in dopaminergic neurons during aging and sporadic Parkinson disease.