In Search of Effective Treatments Targeting α-Synuclein Toxicity in Synucleinopathies: Pros and Cons

Abstract

Parkinson's disease (PD), multiple system atrophy (MSA) and Dementia with Lewy bodies (DLB) represent pathologically similar, progressive neurodegenerative disorders characterized by the pathological aggregation of the neuronal protein α-synuclein. PD and DLB are characterized by the abnormal accumulation and aggregation of α-synuclein in proteinaceous inclusions within neurons named Lewy bodies (LBs) and Lewy neurites (LNs), whereas in MSA α-synuclein inclusions are mainly detected within oligodendrocytes named glial cytoplasmic inclusions (GCIs). The presence of pathologically aggregated α-synuclein along with components of the protein degradation machinery, such as ubiquitin and p62, in LBs and GCIs is considered to underlie the pathogenic cascade that eventually leads to the severe neurodegeneration and neuroinflammation that characterizes these diseases. Importantly, α-synuclein is proposed to undergo pathogenic misfolding and oligomerization into higher-order structures, revealing self-templating conformations, and to exert the ability of "prion-like" spreading between cells. Therefore, the manner in which the protein is produced, is modified within neural cells and is degraded, represents a major focus of current research efforts in the field. Given that α-synuclein protein load is critical to disease pathogenesis, the identification of means to limit intracellular protein burden and halt α-synuclein propagation represents an obvious therapeutic approach in synucleinopathies. However, up to date the development of effective therapeutic strategies to prevent degeneration in synucleinopathies is limited, due to the lack of knowledge regarding the precise mechanisms underlying the observed pathology. This review critically summarizes the recent developed strategies to counteract α-synuclein toxicity, including those aimed to increase protein degradation, to prevent protein aggregation and cell-to-cell propagation, or to engage antibodies against α-synuclein and discuss open questions and unknowns for future therapeutic approaches.

Keywords: autophagy; immunotherapy; propagation; proteasome; protein aggregation; synucleinopathies; therapeutics; α-synuclein.

FIGURE 1

Structure of α-synuclein. The N-terminal domain of α-synuclein is characterized by the presence of repeated lipid-binding sequences and contains the mutation sites linked with familial PD. The central NAC domain is mainly hydrophobic and favors the aggregation process of the protein. The C-terminal acidic tail carries the majority of α-synuclein phosphorylation sites.

FIGURE 2

α-synuclein degradation pathways and reported therapeutic approaches to enhance protein clearance. A schematic representation of the main proteolytic pathways implicated in α-synuclein clearance and the proposed targets for potential therapeutic interventions (highlighted in dark grey and green). Wild type and mutant α-synuclein can undergo both ubiquitin-dependent (A) and ubiquitin-independent (B) degradation via the 20S/26S proteasome. Monomeric wild-type α-synuclein is degraded via CMA, following its binding to the CMA-specific receptor, LAMP2A (C). Molecular upregulation of LAMP2A expression or chemical enhancement of CMA through retinoic acid receptor alpha (RARα) antagonists has been proven successful in alleviating α-synuclein-associated toxicity. Macroautophagy has been also proposed to degrade mutant and aggregated forms of α-synuclein (D). Boosting macroautophagy via mTOR-dependent (rapamycin) or mTOR-independent pharmacological and nutritional modulators (Metformin, Nilotinib, AICAR, Trehalose, Resveratrol, Pomegranate, C1) enhance autophagosome formation, lysosome biogenesis, and lysosome function thus promoting α-synuclein clearance (E). Molecular modulation of macroautophagy via Atg7, Beclin1 or TFEB overexpression is also reported to exert beneficial effects on α-synuclein-related toxicity (E). Lastly, restoration of proper enzymatic activity of GCase has been shown to improve lysosomal function and lessen α-synuclein levels (F).

FIGURE 3

Proposed mechanisms of α-synuclein cell-to-cell propagation and application of candidate therapeutic strategies. A schematic representation of α-synuclein transfer from neurons to neurons (purple) or to oligodendrocytes (blue) via various mechanisms: (A) Neuronal receptors (i.e., LAG3) interact with extracellular α-synuclein and mediate its internalization via endocytosis. Antibodies against these receptors effectively inhibit α-synuclein propagation. (B) Neuronally-derived, free or exosomal-bound α-synuclein enters neighboring neurons or oligodendrocytes. (C) α-synuclein is taken-up by cells via passive diffusion or direct penetration of their plasma membrane. (D) Clathrin- or dynamin- mediated endocytosis is responsible for α-synuclein internalization in neurons and oligodendrocytes. Inhibitors targeting these endocytic pathways have been effectively used. (E) Heparan Sulfate ProteoGlycans (HSPGs) regulate α-synuclein uptake via macropinocytosis in neurons and oligodendrocytes. Disruption of HSPGs by chemical molecules (heparin or chloral hydrate) inhibits α-synuclein uptake by cells. (F) Tunneling nanotubes (thin membranous bridges) have been also proposed as a possible cell-to-cell transmission mechanism of α-synuclein. (G) siRNAs (small interfere RNAs) designed against α-synuclein mRNA are used for the reduction of α-synuclein production as an effective therapeutic strategy. (H) α-synuclein entrance in neuronal or oligodendroglial cells is followed by its aggregation and the spread of α-synuclein pathology (seeding), finally leading to the formation of aberrant protein species. Various antibodies targeting the NAC or the C-terminal region of α-synuclein and chemical molecules and compounds (i.e., NPT200–11, NPT100-18A, NPT088 etc.) inhibiting α-synuclein aggregation have been used to prevent α-synuclein misfolding.

FIGURE 4

Immunotherapy approaches against α-synuclein. (A) Thermodynamically unstable monomers of α-synuclein misfold and aggregate into pathological species. The release of oligomeric and fibrillar α-synuclein into the extracellular space triggers α-synuclein propagation into non-affected cells, microglial activation and neuroinflammation. (B) Active or passive immunization mainly aims to lower the extracellular α-synuclein by microglial-mediated degradation and to prevent pathology propagation via antibody binding on receptors facilitating α-synuclein endocytosis. (C) The effective targeting of intracellular α-synuclein is achieved by antibody fragments paired with signaling peptides for endocytosis, or viral vector-derived antibody constructs, which are expressed within the cells. (D) The engineered intrabodies bind to α-synuclein toxic species and lead them to degradation through proteasomal or autophagic pathways. (E) Antibodies against immune system activation is another immunotherapy approach, which aims to reduce pro-inflammatory cytokine release, enhance anti-inflammatory microglial activity and, by that, prevent α-synuclein pathology progression.