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In patients with Parkinson’s disease (PD), constipation is common, and it appears in a prodromal stage before the hallmark motor symptoms. The present study aimed to investigate whether Velusetrag, a selective 5‑HT4 receptor agonist, may be a suitable candidate to improve intestinal motility in a mouse model of PD. Five months old PrP human A53T alpha-synuclein transgenic mice, which display severe constipation along with decreased colonic cholinergic transmission already at 3 months, were treated daily with the drug for 4 weeks. Velusetrag treatment reduced constipation by significantly stimulating both the longitudinal and circular-driven contractions and improved inflammation by reducing the level of serum and colonic IL-1β and TNFα and by decreasing the number of GFAP-positive glia cells in the colon of treated mice. No downregulation of the 5-HT4 receptor was observed but instead Velusetrag seemed to induce axonal regeneration in the colon, stimulating the AKT pathway. Ultimately, Velusetrag restored a well-balanced intestinal microbial composition comparable to non transgenic mice. Based on these promising data, we are confident that Velusetrag is potentially eligible for clinical studies to treat constipation in PD patients.

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

Parkinson’s Disease (PD) is typically classified as a neurodegenerative disease affecting the motor system. Recent evidence, however, has uncovered the presence of Lewy bodies in locations outside the CNS, in direct contact with the external environment, including the olfactory bulbs and the enteric nervous system. This, combined with the ability of alpha-synuclein (αS) to propagate in a prion-like manner, has supported the hypothesis that the resident microbial community, commonly referred to as microbiota, might play a causative role in the development of PD. In this article, we will be reviewing current knowledge on the importance of the microbiota in PD pathology, concentrating our investigation on mechanisms of microbiota-host interactions that might become harmful and favor the onset of PD. Such processes, which include the secretion of bacterial amyloid proteins or other metabolites, may influence the aggregation propensity of αS directly or indirectly, for example by favoring a pro-inflammatory environment in the gut. Thus, while the development of PD has not yet being associated with a unique microbial species, more data will be necessary to examine potential harmful interactions between the microbiota and the host, and to understand their relevance in PD pathogenesis.

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.

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 (alpha S) is a small protein that self-aggregates into a-helical oligomer species and subsequently into larger insoluble amyloid fibrils that accumulate in intraneuronal inclusions during the development of Parkinson's disease. Toxicity of alpha S 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 alpha S species accumulate, rather than their conformation, might contribute to enhanced toxicity. In line with this observation, we have shown that alpha S oligomers and aggregates are associated with the endoplasmic reticulum/microsomes (ER/M) membrane in vivo and how accumulation of soluble alpha S oligomers at the ER/M level precedes neuronal degeneration in a mouse model of alpha-synucleinopathies. In this paper we took a further step, investigating the biochemical and functional features of alpha S species associated with the ER/M membrane. We found that by comparison with non-microsomal associated alpha S (P10), the ER/M-associated alpha S 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 alpha S species isolated from diseased A53T human alpha S transgenic mice induced neuronal changes in a time- and dose-dependent manner. In fact the addition of small amounts of ER/M-associated alpha S species from diseased mice to primary cultures induced the formation of beads-like structures or strings of fibrous alpha S 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 alpha S high molecular weight (HMW) species. These data provide the first evidence of the existence of two different populations of alpha S HMW species in vivo, putting the spotlight on the association to ER/M membrane as a necessary step for the acquisition of alpha S toxic features.

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.

Chaperone-mediated autophagy controls the degradation of selective cytosolic proteins and may protect neurons against degeneration. In a neuronal cell line, we found that chaperone-mediated autophagy regulated the activity of myocyte enhancer factor 2D (MEF2D), a transcription factor required for neuronal survival. MEF2D was observed to continuously shuttle to the cytoplasm, interact with the chaperone Hsc70, and undergo degradation. Inhibition of chaperone-mediated autophagy caused accumulation of inactive MEF2D in the cytoplasm. MEF2D levels were increased in the brains of α-synuclein transgenic mice and patients with Parkinson's disease. Wild-type α-synuclein and a Parkinson's disease–associated mutant disrupted the MEF2D-Hsc70 binding and led to neuronal death. Thus, chaperone-mediated autophagy modulates the neuronal survival machinery, and dysregulation of this pathway is associated with Parkinson's disease.

The pathophysiological processes that cause Parkinson's disease (PD) affect dopamine neurons residing in the substantia nigra with devastating consequences for normal movement. One important gene involved in both familial and sporadic PD is alpha-synuclein. We have generated three strains of alpha-synuclein transgenic mice to study the pathologic consequences of the targeted expression of mutant or wild-type human alpha-synuclein in a model system. We have analyzed gene expression patterns in these mice using high throughput microarrays in anatomical regions implicated in disease (substantia nigra and brainstem). Our study reveals gene dosage-dependent dysregulation of several genes important for the dopaminergic phenotype in mice over-expressing wild-type human alpha-synuclein in the substantia nigra at time points preceding neuronal cell death. Analysis of mutant alpha-synuclein mice at a time point when pathology is advanced reveals several new candidate genes that may play a role in neuronal demise and/or protein accumulation.

TonEBP [TonE (tonicity-responsive enhancer)-binding protein] is a transcriptional activator of the Rel family like NF-κB (nuclear factor κB) and NFAT (nuclear factor of activated T-cells). TonEBP plays a key role in the protection of cells in the kidney medulla from the deleterious effects of hyperosmolality. This is achieved by enhancing expression of HSP70 (heat-shock protein 70) and other genes whose products drive cellular accumulation of organic osmolytes. TonEBP is stimulated by ambient hypertonicity via multiple pathways that regulate nuclear translocation and transactivation. In the present paper, we report that TonEBP is associated in vivo with RHA (RNA helicase A). The N- and C-termini of RHA bound the E′F loop of the DNA-binding domain of TonEBP. The interaction was not affected by DNA binding or dimerization of TonEBP. Overexpression of RHA inhibited the activity of TonEBP; however, catalytic activity of RHA was dispensable for the inhibition. When the ambient tonicity was raised, the TonEBP–RHA interaction decreased, suggesting that dissociation of RHA is a pathway to stimulate TonEBP. We conclude that the E′F loop of TonEBP interacts with RHA like NFAT and NF-κB interact with AP1 (activator protein 1) and the high-mobility group protein HMG-I(Y) respectively. While RHA interacts with and stimulates other transcription factors such as CREB (cAMP-response-element-binding protein), NF-κB and mineralocorticoid receptor, it inhibits TonEBP.

In Parkinson's disease (PD) and other α-synucleinopathies, prefibrillar α-synuclein (αS) oligomer is implicated in the pathogenesis. However, toxic αS oligomers observed usingin vitrosystems are not generally seen to be associated with α-synucleinopathyin vivo. Thus, the pathologic significance of αS oligomers to αS neurotoxicity is unknown. Herein, we show that, αS that accumulate within endoplasmic reticulum (ER)/microsome forms toxic oligomers in mouse and human brain with the α-synucleinopathy. In the mouse model of α-synucleinopathy, αS oligomers initially form before the onset of disease and continue to accumulate with the disease progression. Significantly, treatment of αS transgenic mice with Salubrinal, an anti-ER stress compound that delays the onset of disease, reduces ER accumulation of αS oligomers. These results indicate that αS oligomers with toxic conformation accumulate in ER, and αS oligomer-dependent ER stress is pathologically relevant for PD.

Accumulation of misfolded α-synuclein (αS) is mechanistically linked to neurodegeneration in Parkinson's disease (PD) and other α-synucleinopathies. However, how αS causes neurodegeneration is unresolved. Because cellular accumulation of misfolded proteins can lead to endoplasmic reticulum stress/unfolded protein response (ERS/UPR), chronic ERS could contribute to neurodegeneration in α-synucleinopathy. Using the A53T mutant human αS transgenic (A53TαSTg) mouse model of α-synucleinopathy, we show that disease onset in the αS Tg model is coincident with induction of ER chaperones in neurons exhibiting αS pathology. However, the neuronal ER chaperone induction was not accompanied by the activation of phospho-eIF2α, indicating that α-synucleinopathy is associated with abnormal UPR that could promote cell death. Induction of ERS/UPR was associated with increased levels of ER/microsomal (ER/M) associated αS monomers and aggregates. Significantly, human PD cases also exhibit higher relative levels of ER/M αS than the control cases. Moreover, αS interacts with ER chaperones and overexpression of αS sensitizes neuronal cells to ERS-induced toxicity, suggesting that αS may have direct impact on ER function. This view is supported by the presence of ERS-activated caspase-12 and the accumulation of ER-associated polyubiquitin. More important, treatment with Salubrinal, an anti-ERS compound, significantly attenuates disease manifestations in both theA53TαSTg mouse model and the adeno-associated virus-transduced rat model ofA53TαS-dependent dopaminergic neurodegeneration. Our data indicate that the accumulation αS within ER leads to chronic ER stress conditions that contribute to neurodegeneration in α-synucleinopathies. Attenuating chronic ERS could be an effective therapy for PD and other α-synucleinopathies.

Abnormal biology of α-synuclein (α-Syn) is directly implicated in the pathogenesis of Parkinson's disease and other α-synucleinopathies. Herein, we demonstrate that C-terminally truncated α-Syn (α-SynΔC), enriched in the pathological α-Syn aggregates, is normally generated from full-length α-Syn independent of α-Syn aggregation in brains and in cultured cells. The accumulation of α-SynΔC is enhanced in neuronal cells as compared with nonneuronal cells. Significantly, the expression of familial Parkinson's disease-linked mutant α-Syn is associated with the enhanced cellular accumulation of α-SynΔC. Moreover, substoichiometric amounts of α-SynΔC enhance the in vitro aggregation of the more abundant full-length α-Syn. Finally, cases of α-synucleinopathy exhibit increases in the total soluble α-Syn and a higher proportion of soluble α-SynΔC, a condition favoring the aggregation of α-Syn. Collectively, our results indicate that the biology behind the generation and accumulation of α-SynΔC is likely to have relevance for the initiation and the progression of α-Syn aggregation in vivo .

Tonicity-responsive enhancer binding protein (TonEBP), also known as NFAT5, belongs to the Rel family of transcriptional activators. In the kidney medulla and thymus, TonEBP plays a major role in protecting renal cells and T cells from the deleterious effects of ambient hypertonicity. TonEBP is stimulated by hypertonicity via several pathways: increased expression of protein, nuclear translocation, and increased transactivation. In this study, we identified five domains of TonEBP involved in transactivation. The two conserved glutamine repeats were not involved in transactivation. There were three activation domains that could stimulate transcription independently. In addition, there were two modulation domains that potentiated the activity of the activation domains. One of the activation domains is unique to a splice isoform that is more active than others, indicating that alternative splicing can affect the activity of TonEBP. Another activation domain and one of the modulation domains were stimulated by hypertonicity. All the five domains acted in synergy in every combination. Although overall phosphorylation of TonEBP increased in response to hypertonicity, phosphorylation of the activation and modulation domains did not increase in isolation. In sum, TonEBP possesses far more elaborate domains involved in transactivation compared with other Rel proteins.

Human umbilical vein endothelial cells transport arginine through two Na(+)-independent systems. System y(+)L is insensitive to N-ethylmaleimide (NEM), inhibited by L-leucine in the presence of Na(+), and referable to the expression of SLC7A6/y(+)LAT2, SLC7A7/y(+)LAT1, and SLC3A2/4F2hc. System y(+) is referable to the expression of SLC7A1/CAT1 and SLC7A2/CAT2B. Tumor necrosis factor-alpha (TNF-alpha) and bacterial lipopolysaccharide induce a transient stimulation of arginine influx and efflux through system y(+). Increased expression of SLC7A2/CAT2B is detectable from 3 h of treatment, while SLC7A1 expression is inhibited at later times of incubation. System y(+)L activity and expression remain unaltered. Nitric oxide synthase type 2 mRNA is not detected in the absence or presence of TNF-alpha, while the latter condition lowers nitric oxide synthase type 3 expression at the mRNA and the protein level. Nitrite accumulation is comparable in cytokine-treated and control cells up to 48 h of treatment. It is concluded that modulation of endothelial arginine transport by TNF-alpha or lipopolysaccharide occurs exclusively through changes in CAT2B and CAT1 expression and is dissociated from stimulation of nitric oxide production. Human umbilical vein endothelial cells transport arginine through two Na+-independent systems. System y+L is insensitive to N-ethylmaleimide (NEM), inhibited by L-leucine in the presence of Na+, and referable to the expression of SLC7A6/y+LAT2, SLC7A7/y+LAT1, and SLC3A2/4F2hc. System y+ is referable to the expression of SLC7A1/CAT1 and SLC7A2/CAT2B. Tumor necrosis factor-α (TNF-α) and bacterial lipopolysaccharide induce a transient stimulation of arginine influx and efflux through system y+. Increased expression of SLC7A2/CAT2B is detectable from 3 h of treatment, while SLC7A1 expression is inhibited at later times of incubation. System y+L activity and expression remain unaltered. Nitric oxide synthase type 2 mRNA is not detected in the absence or presence of TNF-α, while the latter condition lowers nitric oxide synthase type 3 expression at the mRNA and the protein level. Nitrite accumulation is comparable in cytokine-treated and control cells up to 48 h of treatment. It is concluded that modulation of endothelial arginine transport by TNF-α or lipopolysaccharide occurs exclusively through changes in CAT2B and CAT1 expression and is dissociated from stimulation of nitric oxide production.