To understand the development and progression of a human disease, an effective model that combines the genetic underpinnings with a similar, if not identical, phenotypic output is required. The appreciable gaps in our understanding of the mechanisms of PD initiation and progression, along with other sporadic and genetic neurodegenerative diseases, can be traced to the current lack of accurate models of these complex human diseases. The standard process for modeling human disease with genetic origins has been to overexpress the human gene of interest in model organisms (from yeast, to drosophila, to mouse, to non-human primate) and then observe the cellular and protein response. Perturbations that appear phenotypic are used as the fitness function for subsequent drug screening or therapeutic target selection. These studies have provided some important advances for a large spectrum of human disease, and have been instrumental in our basic understanding of neurodegenerative diseases [33
However, the use of these transgenic models have failed to develop an accurate portrait of the underlying disease mechanisms. This unfortunate result should not be surprising. While overexpression of human genes in model organisms may constitute an accurate representation of the underlying human pathogenesis for some diseases, in complex neurodegenerative diseases that involve endogenously expressed human proteins, basic cellular machinery such as transcriptional feedback, and stochastic balance of the proteasomal pathway are bound to play significant roles. These two fundamental cellular processes are greatly compromised in overexpression organism models: transcriptional feedback is impossible when proteins are expressed on a non-native promoter, or even a native promoter in a different location in the genome, where epigenetics and other sequence neighbor effects will undoubtedly play a role in expression. The result is that mechanistic studies of neurodegeneration have been thwarted by an inability to study the cellular processes in susceptible human neurons that had an endogenous predisposition to the disease.
The lack of studies employing human cells to investigate human neurodegenerative disease is not due to a lack of interest. PD is challenging to study due to the inaccessibility of affected human midbrain dopaminergic (mDA) neurons and a scarcity of animal models that mimic the key disease features. The transgenic animals and cellular models expressing known PD-associated genes have provided important insights into the pathogenesis of the disease; however, it has been difficult to demonstrate that the implicated mechanisms are also present in neurons from affected individuals.
Previous genetic studies have implicated several potential mechanisms but the pathogenesis of PD remains largely obscure. In most PD patients, Lewy bodies and Lewy neurites form in both CNS and autonomic PNS neurons. These large intracellular proteinaceous inclusions are rich in α-synuclein and ubiquitin, an observation that suggests a role for α-synuclein and the proteasome in the molecular development of sporadic and inherited PD [40
]. However, not all patients with PD develop Lewy bodies. Although mutations in LRRK2
are commonly associated with PD and Lewy body pathology, some of these patients do not exhibit a Lewy phenotype. Therefore, it is thought that protein aggregration may actually be a disease modifier rather than direct cause [41
]. Methods to identify patients with a well-defined aggregation phenotype may be useful in categorizing disease, as well as in correctly targeting disease-modifying interventions to an appropriate class of patients.
As discussed, autosomal-dominant forms of PD have been documented in families with SNCA missense mutations or gene duplication/triplications. α-Synuclein protein aggregation and markers of cellular stress are also seen in other neurodegenerative disorders, including multiple system atrophy, Bradbury-Eggleston syndrome, and dementia with Lewy bodies.
The selective degeneration of neural subtypes in each disease has since been linked to the resulting cornucopia of observed clinical manifestations. However, recent evidence suggests that the true culprit of cellular toxicity may be the soluble cytoplasmic oligomeric α-synuclein protein, whereas the large insoluble protein aggregates may in fact represent a cellular defense mechanism in which the cell sequesters cytotoxic-soluble oligomeric proteins into insoluble inclusion bodies [42
Attempting to replicate the cardinal phenotypic features of PD, researchers have made use of animal models of dopaminergic neurotoxicity, and transgenic models of the familial PD-causing genes. In drosophila, overexpression of SNCA leads to age-dependent dopaminergic neuron degeneration. Also, transgenic mice overexpressing mouse and human SNCA exhibit pathological phenotypes—inclusion body formation in some CNS cells, slight impairment in motor control, and loss of dopaminergic neuron terminals in the basal ganglia. These results from transgenic models support a causal role for α-synuclein in the development of PD [43
]. Furthermore, the experiments using LRRK2-G2019S
transgenic mouse models demonstrated age-dependent and levodopa-responsive slowness of movement associated with diminished dopamine release and axonal pathology [46
Taking together this body of evidence and the known inherited forms of PD, one can construct a basic PD model: α-synuclein begins to aggregate, through some innate mechanism, or induced by other factors; the cell recognizes the buildup of cytosolic α-synuclein levels and attempts to degrade the proteins by ubiquitination, and subsequent shuttling to a proteasome for degradation. In the diseased brain, any one of the following processes could be disturbed: the innate dynamic aggregation of α-synuclein, the regulation of α-synuclein expression level, ubiquitination of α-synuclein, and degradation of α-synuclein degradation. Interestingly, Lewy bodies generally contain high concentrations of ubiquitin, suggesting unfruitful attempts by the cellular proteasomal pathway to degrade α-synuclein despite substantial poly-ubiquitination.
In sum, while it has been shown that overexpression of α-synuclein transgenes leads to protein aggregation in normal cells, the study of native processes leading to aggregation in affected individuals has been hindered by the inaccessibility of human neurons in vivo, the limitations inherent in studying postmortem samples from PD patients, and the inability to accurately recapitulate human disease in transgenic models [47