In virto model systems for alpha-synuclein

Cell-culture models of a-synuclein provide a valuable route for studying the physiologic and pathologic functions of the protein because they would be potentially amenable to high throughput translation to identify therapeutic compounds. a- Syn can be highly overexpressed in various cell lines and primary neuronal culture systems. Expression of a-syn containing a PD-associated point mutation in PC12 cells leads to loss of dopaminergic release, alteration of the ubiquitin dependent degradation system, and autophagic-dependent cell death. Overexpression of a-syn in H4-glioma cells results in an upregulation of markers associated with toxicity. Delivery of a-syn via HSV-1 transduction in mesencephalic
primary cultures likewise results in enhanced toxicity in infected cells. However, the goal of achieving a robust model of a-syn–dependent cell death in cultured cells amenable to translation to high-throughput screening remains an elusive goal.
Most described a-syn–overexpression systems in mammalian cells result in very mild to no significant changes in markers of toxicity associated with wild-type a-syn expression. Some culture systems demonstrate neuroprotective mechanisms due to a-syn expression. a-Syn expressed in NT-2D1 (human teratocarcinoma cell line) and SK-MC (human neuroblastoma cell line) cells delays cell death induced by serum withdrawal, but the effect is reversed via MPPþ (1-methyl-4-phenylpyridinium) exposure. In lieu of overt changes in cell-death markers associated with a-syn expression, numerous studies have described potential early phenotypes with possible relevance to pathogenesis. A kinetic basis for intracellular accumulation has been demonstrated by
overexpressing Flag- and His-tagged versions of a-syn in PC12 and SH-SY5Y cells. Demonstration of a-syn expression and oligomeric intermediates in living cells through bimolecular fluorescence complementation may also provide insight into a-syn mechanisms. Because overt cell death caused by a-syn expression has been difficult to achieve in mammalian cells, the future of in vitro model systems will likely involve phenotypes outside of toxicity but focused on particular aspects of a-syn conformation, association, or localization. The relevance of data developed in vitro will require confirmation in successful in vivo model systems.

Alpha synuclein Target Validation

The majority of therapies for human disease involve the inhibition of a particular target important for pathogenesis. Many clinical trials fail because the target itself is not a critical component of disease. As a result, pharmaceutical companies
now demand more-stringent target-validation studies. Pathologic evidence from human PD tissue nominating a-syn as the primary agent of pathogenesis is circumstantial, whereas
human genetic studies firmly place the causative basis with asyn, but only in a very small proportion of familial and early onset- disease cases. The idea that a-syn is a robust therapeutic target in PD is provocative but nonetheless requires extensive
proof from target-validation studies. More than a decade of research centered on a-syn highlights outstanding questions through the descriptions of numerous model systems.

Alpha synuclein aggregation: mechanism and role in PD

As a-syn is the most abundant protein composing the proteinaceous aggregates that define PD on a pathologic level, intense efforts revolve around understanding a-syn
accumulation and aggregation. a-Syn may assume oligomeric species through unknown mechanisms, and higher-order a-syn structures usually correlate with a-syn–dependent toxicity in cells (Fig). The specific conformational entities responsible
for protection, toxicity, and / or aggregation remain elusive. Factors known to modify a-syn aggregation and/or oligomerization include alterations of the primary amino acid sequence (e.g., PD-associated mutations); c-terminal truncations,
interactions with metal ions; interactions with Ab peptide; interactions with chaperone proteins such as Hsp70; interactions with apolipoprotein E, as well as
neurotoxins, pesticides, and herbicides; organic solvents; tyrosine nitration; phosphorylation; methionine oxidation; monoubiquitination; interaction with
polyanions and polycations, oxidative dimmers, and oligomers; histones; transglutaminase;and other protein–protein interactions.
As in other neurodegenerative disorders and diseases associated with protein aggregation, the role of organized inclusion bodies localized to disease-associated regions in PD affected tissue remains hotly debated. On the one hand, the
inclusion-body organelles may serve to sequester molecules that, for whatever reason, can no longer route through normal metabolism that would otherwise cause cellular dysfunction.
Experimental evidence supports this notion, in which the lack of a-syn–inclusion formation associates with toxicity. In contrast, the overt formation of inclusions may represent the toxic insult itself composed of molecules that were otherwise
nontoxic until association with the inclusion. In the first case, promoting inclusion bodies should facilitate neuroprotection. In the second case, inhibiting inclusion bodies would promote cell toxicity. Both situations may be true,
depending on the particular component of the inclusion body in question; for example, sequestration of toxic a-syn species into inclusion bodies may represent cytoprotection, with the cost of sequestering and inactivating other molecules necessary for normal function.
Therapeutically oriented efforts should focus on farther upstream events as opposed to modifying preexisting inclusion bodies, although this may be a moot point because model systems that demonstrate inclusions with morphologic similarity to Lewy bodies have yet to be developed. One major obstacle to understanding Lewy bodies is the failure to recapitulate the basic morphologic characteristics of Lewy bodies
in the context of relevant model systems. Although several model systems describe a-syn– and ubiquitin-positive aggregations in some fraction of cells, none can be
considered similar to the highly ordered structures found in PD tissue.
As a potential therapeutic target, the accumulation of a-syn into insoluble protein inclusions seems an important event in pathogenesis. A strong case can be made for therapeutically promoting inclusion formation in disease to protect cells from
more-soluble toxic species, as well as therapeutic approaches to dissolve inclusions to relieve the cells of a deleterious organelle that disrupts normal function. The dichotomy may not be resolved in the near future and hinders a-syn– antiaggregation strategies as a viable therapeutic approach.

Alpha synuclein: Primary Structure

a-Syn is one of the most abundant ‘‘natively unfolded’’(intrinsically disordered) proteins that likely have different morphologies, including protofibrils, oligomers, and fibrils. Under physiologic conditions, a-syn has little or no ordered structure and possesses noteworthy conformational plasticity. The primary structure of a-syn is characterized as follows: (a) Residues 1-60 constitute the N-terminal domain
encompassing the conserved imperfect hexameric repeats (KTKEGV) and includes three missense mutations associated with early-onset familial PD (A30P, E46K, and A53T). The conserved 11-residue (XKTKEGVXXXA, X = any amino acid)repeat forms amphipathic secondary a-helical structures on binding to acidic phospholipid membranes, typical of the lipid-binding domains of apolipoproteins; (b) the central region (residues 61 to 95) is composed of an extremely hydrophobic, highly aggregation-prone NAC (nonamyloid component) sequence; and (c) the hydrophilic C-terminal region consists of highly acidic residues glutamic and aspartic acid, as well as proline, and is responsible for chaperone function.