Toxic properties of microsome-associated alpha-synuclein species in mouse primary neurons.

α-synuclein (αS) is a small protein that self-aggregates into α-helical oligomer species and subsequently into larger insoluble amyloid fibrils that accumulate in intraneuronal inclusions during the development of Parkinson's disease. Toxicity of αS oligomers and fibrils has been long debated and more recent data are suggesting that both species can induce neurodegeneration. However while most of these data are based on differences in structure between oligomer and aggregates, often preassembled in vitro, the in vivo situation might be more complex and subcellular locations where αS species accumulate, rather than their conformation, might contribute to enhanced toxicity. In line with this observation, we have shown that αS oligomers and aggregates are associated with the endoplasmic reticulum/microsomes (ER/M) membrane in vivo and how accumulation of soluble αS oligomers at the ER/M level precedes neuronal degeneration in a mouse model of α-synucleinopathies. In this paper we took a further step, investigating the biochemical and functional features of αS species associated with the ER/M membrane. We found that by comparison with non-microsomal associated αS (P10), the ER/M-associated αS pool is a unique population of oligomers and aggregates with specific biochemical traits such as increased aggregation, N- and C-terminal truncations and phosphorylation at serine 129. Moreover, when administered to murine primary neurons, ER/M-associated αS species isolated from diseased A53T human αS transgenic mice induced neuronal changes in a time- and dose-dependent manner. In fact the addition of small amounts of ER/M-associated αS species from diseased mice to primary cultures induced the formation of beads-like structures or strings of fibrous αS aggregates along the neurites, occasionally covering the entire process or localizing at the soma level. By comparison treatment with P10 fractions from the same diseased mice resulted in the formation of scarce and small puncta only when administered at high amount. Moreover, increasing the amount of P100/M fractions obtained from diseased and, more surprisingly, from presymptomatic mice induced a significant level of neuronal death that was prevented when neurons were treated with ER/M fractions immunodepleted of αS high molecular weight (HMW) species. These data provide the first evidence of the existence of two different populations of αS HMW species in vivo, putting the spotlight on the association to ER/M membrane as a necessary step for the acquisition of αS toxic features.

Alpha-Synuclein FRET Biosensors Reveal Early Alpha-Synuclein Aggregation in the Endoplasmic Reticulum.

Endoplasmic reticulum (ER) dysfunction is important for alpha-synuclein (αS) acquired toxicity. When targeted to the ER in SH-SY5Y cells, transient or stable expression of αS resulted in the formation of compact αS-positive structures in a small subpopulation of cells, resembling αS inclusions. Thus, because of the limitations of immunofluorescence, we developed a set of αS FRET biosensors (AFBs) able to track αS conformation in cells. In native conditions, expression in i36, a stable cell line for ER αS, of intermolecular AFBs, reporters in which CFP or YFP has been fused with the C-terminal of αS (αS-CFP/αS-YFP), resulted in no Förster resonance energy transfer (FRET), whereas expression of the intramolecular AFB, a probe obtained by fusing YFP and CFP with αS N- or C- termini (YFP-αS-CFP), showed a positive FRET signal. These data confirmed that αS has a predominantly globular, monomeric conformation in native conditions. Differently, under pro-aggregating conditions, the intermolecular AFB was able to sense significantly formation of αS oligomers, when AFB was expressed in the ER rather than ubiquitously, suggesting that the ER can favor changes in αS conformation when aggregation is stimulated. These results show the potential of AFBs as a new, valuable tool to track αS conformational changes in vivo.

Constipation, deficit in colon contractions and alpha-synuclein inclusions within the colon precede motor abnormalities and neurodegeneration in the central nervous system in a mouse model of alpha-synucleinopathy.

BACKGROUND: Gastrointestinal dysfunction can affect Parkinson's disease (PD) patients long before the onset of motor symptoms. However, little is known about the relationship between gastrointestinal abnormalities and the development of PD. Contrary to other animal models, the human A53T alpha-synuclein (αS) transgenic mice, Line G2-3, develops αS-driven neurological and motor impairments after 9 months of age, displaying a long presymptomatic phase free of central nervous system (CNS) dysfunction. METHODS: To determine whether this line can be suitable to study constipation as it occurs in prodromal PD, gastrointestinal functionality was assessed in young mice through a multidisciplinary approach, based on behavioral and biochemical analysis combined with electrophysiological recordings of mouse intestinal preparations. RESULTS: We found that the A53T αS mice display remarkable signs of gastrointestinal dysfunction that precede motor abnormalities and αS pathology in the CNS by at least 6 months. Young αS mice show a drastic delay in food transit along the gastrointestinal tract, of almost 2 h in 3 months old mice that increased to more than 3 h at 6 months. Such impairment was associated with abnormal formation of stools that resulted in less abundant but longer pellets excreted, suggesting a deficit in the intestinal peristalsis. In agreement with this, electrically evoked contractions of the colon, but not of the ileum, showed a reduced motor response in both longitudinal and circular muscle layers in αS mice already at 3 months of age, that was mainly due to an impaired cholinergic transmission of the underlying enteric nervous system. Interestingly, the presence of insoluble and aggregated αS was found in enteric neurons in both myenteric and submucosal plexi only in the colon of 3 months old αS mice, but not in the small intestine, and exacerbated with age, mimicking the increase in transit delay and the contraction deficit showed by behavioral and electrical recordings data. CONCLUSIONS: Gastrointestinal dysfunction in A53T αS mice represents an early sign of αS-driven pathology without concomitant CNS involvement. We believe that this model can be very useful to study disease-modifying strategies that could extend the prodromal phase of PD and halt αS pathology from reaching the brain.

Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation.

For years, the inability of replicating formation of insoluble alpha-synuclein (αS) inclusions in cell cultures has been a great limitation in the study of αS aggregation in Parkinson's Disease (PD). Recently, the development of new animal models through the exogenous inoculation of brain extracts from diseased αS transgenic mice or PD patients has given new hopes to the possibility of creating more adequate cell models of αS aggregation. Unfortunately, when it comes to cells in cultures, administration of raw brain extracts has not proven as successful as in mice and the source of choice of exogenous aggregates is still in vitro preformed αS fibrils. We have developed a method to induce the formation of intracellular αS inclusions in primary neurons through the exogenous administration of native microsomes-associated αS aggregates, a highly toxic αS species isolated from diseased areas of transgenic mice. This fraction of αS aggregates that is associated with the microsomes vesicles, is efficiently internalized and induces the formation of intracellular inclusions positive for aggregated and phosphorylated αS. Compared to in vitro-preformed fibrils which are made from recombinant αS, our method is faster and guarantees that the pathogenic seeding is made with authentic αS aggregates extracted from diseased animal models of PD, mimicking more closely the type of inclusions obtained in vivo. As a result, availability of tissues rich in αS inclusions is mandatory. We believe that this method will provide a versatile cell-based model to study the microscopic aspects of αS aggregation and the related cellular pathophysiology in vivo and will be a starting point for the creation of more accurate and sophisticated cell paradigm of PD.

Subcellular localization of alpha-synuclein aggregates and their interaction with membranes.

For more than a decade numerous evidence has been reported on the mechanisms of toxicity of α-synuclein (αS) oligomers and aggregates in α-synucleinopathies. These species were thought to form freely in the cytoplasm but recent reports of αS multimer conformations when bound to synaptic vesicles in physiological conditions, have raised the question about where αS aggregation initiates. In this review we focus on recent literature regarding the impact on membrane binding and subcellular localization of αS toxic species to understand how regular cellular function of αS contributes to pathology. Notably αS has been reported to mainly associate with specific membranes in neurons such as those of synaptic vesicles, ER/Golgi and the mitochondria, while toxic species of αS have been shown to inhibit, among others, neurotransmission, protein trafficking and mitochondrial function. Strategies interfering with αS membrane binding have shown to improve αS-driven toxicity in worms and in mice. Thus, a selective membrane binding that would result in a specific subcellular localization could be the key to understand how aggregation and pathology evolves, pointing out to αS functions that are primarily affected before onset of irreversible damage.