FTY720 Nanoformulation Induces O-GlcNacylation of Synuclein to Alleviate Synucleinopathy.

The post-translational modification and aggregation of alpha-synuclein are one of the major causes of Parkinson's disease (PD) regulation. In that, the phosphorylation and nitration of synuclein elevate the aggregation, while O-GlcNacylation prevents the aggregation of synuclein. The inhibition of synuclein aggregation directs the development of PD therapy. The endowed O-GlcNacylation of synuclein could be a promising strategy to inhibit synucleinopathy. Therefore, the neuroprotective chitosan-based FTY720 nanoformulation, PP2A (Protein phosphatase 2) activator has been employed to evaluate the PP2A role in the O-GlcNacylation of synuclein in an in vivo PD model. The neuroprotective effect of our nanoformulation is attributed to the upregulation of tyrosine hydroxylase (TH), the PD therapeutic target, with behavioral improvement in animals against rotenone-induced PD deficits. The neuroprotective molecular insights revealed the camouflaged role of PP2A by endowing the OGT activity that induces O-GlcNacylation of synuclein in the reduction of synucleinopathy.

REM behavior disorder: When Parkinson's disease meets Morpheus.

Rapid eye movement (REM) sleep behavior disorder (RBD) is a parasomnia characterized by the absence of normal muscle atonia during REM sleep, resulting in excessive motor activity while dreaming. RBD can be classified as isolated which is the strongest clinical marker of prodromal synucleinopathy, or secondary, associated with other neurological diseases, mainly Parkinson's disease (PD) and dementia with Lewy bodies. The diagnosis of RBD must be systematically documented by a video polysomnography in the case of isolated RBD. PD associated with RBD may represent a distinct phenotype compared to PD without RBD, indicating a more severe and widespread synucleinopathy. Clinically, it is associated with poorer motor and cognitive performance, more severe non-motor symptoms, and faster disease progression. Imaging studies have revealed broader brain damage and significant alterations in cerebral metabolism and neurotransmission in PD patients with RBD. The management of RBD involves safety precautions and pharmacotherapy. Safety measures aim to minimize the risk of injury during RBD episodes and include creating a safe sleeping environment and separating the patient from their bed partner if necessary. Pharmacotherapy options include clonazepam and melatonin. Clonazepam must be cautiously prescribed in older patients due to potential side effects.

Repositioning doxycycline for treating synucleinopathies: Evidence from a pre-clinical mouse model.

BACKGROUND AND PURPOSE: Parkinson's disease remains orphan of valuable therapies capable to interfere with the disease pathogenesis despite the large number of symptomatic approaches adopted in clinical practice to manage this disease. Treatments simultaneously affecting α-synuclein (α-syn) oligomerization and neuroinflammation may counteract Parkinson's disease and related disorders. Recent data demonstrate that Doxycycline, a tetracycline antibiotic, can inhibit α-syn aggregation as well as neuroinflammation. We herein investigate, for the first time, the potential therapeutic properties of Doxy in a human α-syn A53T transgenic Parkinson's disease mouse model evaluating behavioural, biochemical and histopathological parameters. EXPERIMENTAL APPROACH: Human α-syn A53T transgenic mice were treated with Doxycycline (10 mg/kg daily ip) for 30 days. The effect of treatment on motor, cognitive and daily live activity performances were examined. Neuropathological and neurophysiological parameters were assessed through immunocytochemical, electrophysiological and biochemical analysis of cerebral tissue. KEY RESULTS: Doxy treatment abolished cognitive and daily life activity deficiencies in A53T mice. The effect on cognitive functions was associated with neuroprotection, inhibition of α-syn oligomerization and gliosis both in the cortex and hippocampus. Doxy treatment restored hippocampal long-term potentiation in association with the inhibition of pro-inflammatory cytokines expression. Moreover, Doxy ameliorated motor impairment and reduced striatal glial activation in A53T mice. CONCLUSIONS AND IMPLICATIONS: Our findings promote Doxy as a valuable multi-target therapeutic approach counteracting both symptoms and neuropathology in the complex scenario of α-synucleinopathies.

The small aromatic compound SynuClean-D inhibits the aggregation and seeded polymerization of multiple α-synuclein strains.

Parkinson's disease is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra, as well as the accumulation of intraneuronal proteinaceous inclusions known as Lewy bodies and Lewy neurites. The major protein component of Lewy inclusions is the intrinsically disordered protein α-synuclein (α-Syn), which can adopt diverse amyloid structures. Different conformational strains of α-Syn have been proposed to be related to the onset of distinct synucleinopathies; however, how specific amyloid fibrils cause distinctive pathological traits is not clear. Here, we generated three different α-Syn amyloid conformations at different pH and salt concentrations and analyzed the activity of SynuClean-D (SC-D), a small aromatic molecule, on these strains. We show that incubation of α-Syn with SC-D reduced the formation of aggregates and the seeded polymerization of α-Syn in all cases. Moreover, we found that SC-D exhibited a general fibril disaggregation activity. Finally, we demonstrate that treatment with SC-D also reduced strain-specific intracellular accumulation of phosphorylated α-Syn inclusions. Taken together, we conclude that SC-D may be a promising hit compound to inhibit polymorphic α-Syn aggregation.

Effect of Docosahexaenoic Acid (DHA) at the Enteric Level in a Synucleinopathy Mouse Model.

The aggregation of alpha-synuclein protein (αSyn) is a hallmark of Parkinson's disease (PD). Considerable evidence suggests that PD involves an early aggregation of αSyn in the enteric nervous system (ENS), spreading to the brain. While it has previously been reported that omega-3 polyunsaturated fatty acids (ω-3 PUFA) acts as neuroprotective agents in the brain in murine models of PD, their effect in the ENS remains undefined. Here, we studied the effect of dietary supplementation with docosahexaenoic acid (DHA, an ω-3 PUFA), on the ENS, with a particular focus on enteric dopaminergic (DAergic) neurons. Thy1-αSyn mice, which overexpress human αSyn, were fed ad libitum with a control diet, a low ω-3 PUFA diet or a diet supplemented with microencapsulated DHA and then compared with wild-type littermates. Our data indicate that Thy1-αSyn mice showed a lower density of enteric dopaminergic neurons compared with non-transgenic animals. This decrease was prevented by dietary DHA. Although we found that DHA reduced microgliosis in the striatum, we did not observe any evidence of peripheral inflammation. However, we showed that dietary intake of DHA promoted a build-up of ω-3 PUFA-derived endocannabinoid (eCB)-like mediators in plasma and an increase in glucagon-like peptide-1 (GLP-1) and the redox regulator, Nrf2 in the ENS. Taken together, our results suggest that DHA exerts neuroprotection of enteric DAergic neurons in the Thy1-αSyn mice, possibly through alterations in eCB-like mediators, GLP-1 and Nrf2.

Preclinical pharmacology of glucosylceramide synthase inhibitor venglustat in a GBA-related synucleinopathy model.

Mutations in GBA, the gene encoding the lysosomal enzyme glucocerebrosidase (GCase), represent the greatest genetic risk factor for developing synucleinopathies including Parkinson's disease (PD). Additionally, PD patients harboring a mutant GBA allele present with an earlier disease onset and an accelerated disease progression of both motor and non-motor symptoms. Preclinical studies in mouse models of synucleinopathy suggest that modulation of the sphingolipid metabolism pathway via inhibition of glucosylceramide synthase (GCS) using a CNS-penetrant small molecule may be a potential treatment for synucleinopathies. Here, we aim to alleviate the lipid storage burden by inhibiting the de novo synthesis of the primary glycosphingolipid substrate of GCase, glucosylceramide (GlcCer). We have previously shown that systemic GCS inhibition reduced GlcCer and glucosylsphingosine (GlcSph) accumulation, slowed α-synuclein buildup in the hippocampus, and improved cognitive deficits. Here, we studied the efficacy of a brain-penetrant clinical candidate GCS inhibitor, venglustat, in mouse models of GBA-related synucleinopathy, including a heterozygous Gba mouse model which more closely replicates the typical GBA-PD patient genotype. Collectively, these data support the rationale for modulation of GCase-related sphingolipid metabolism as a therapeutic strategy for treating GBA-related synucleinopathies.

Alpha-Synuclein: Mechanisms of Release and Pathology Progression in Synucleinopathies.

The accumulation of misfolded alpha-synuclein (aSyn) throughout the brain, as Lewy pathology, is a phenomenon central to Parkinson's disease (PD) pathogenesis. The stereotypical distribution and evolution of the pathology during disease is often attributed to the cell-to-cell transmission of aSyn between interconnected brain regions. The spreading of conformationally distinct aSyn protein assemblies, commonly referred as strains, is thought to result in a variety of clinically and pathologically heterogenous diseases known as synucleinopathies. Although tremendous progress has been made in the field, the mechanisms involved in the transfer of these assemblies between interconnected neural networks and their role in driving PD progression are still unclear. Here, we present an update of the relevant discoveries supporting or challenging the prion-like spreading hypothesis. We also discuss the importance of aSyn strains in pathology progression and the various putative molecular mechanisms involved in cell-to-cell protein release. Understanding the pathways underlying aSyn propagation will contribute to determining the etiology of PD and related synucleinopathies but also assist in the development of new therapeutic strategies.

Clinical Trials for Neurogenic Orthostatic Hypotension: A Comprehensive Review of Endpoints, Pitfalls, and Challenges.

Neurogenic orthostatic hypotension (nOH) is among the most debilitating nonmotor features of patients with Parkinson's disease (PD) and other synucleinopathies. Patients with PD and nOH generate more hospitalizations, make more emergency room visits, create more telephone calls/mails to doctors, and have earlier mortality than those with PD but without nOH. Overall, the health-related cost in patients with PD and OH is 2.5-fold higher compared with patients with PD without OH. Hence, developing effective therapies for nOH should be a research priority. In the last few decades, improved understanding of the pathophysiology of nOH has led to the identification of therapeutic targets and the development and approval of two drugs, midodrine and droxidopa. More effective and safer therapies, however, are still needed, particularly agents that could selectively increase blood pressure only in the standing position because supine hypertension is the main limitation of available drugs. Here we review the design and conduct of nOH clinical trials in patients with PD and other synucleinopathies, summarize the results of the most recently completed and ongoing trials, and discuss challenges, bottlenecks, and potential remedies.

Deciphering the robustness of pyrazolo-pyridine carboxylate core structure-based compounds for inhibiting α-synuclein in transgenic C. elegans model of Synucleinopathy.

Parkinson's disease (PD), a calamitous neurodegenerative disorder with no cure till date, is closely allied with the misfolding and aggregation of α-Synuclein (α -Syn). Inhibition of α-Syn aggregation is one of the optimistic approaches for the treatment for PD. Here, we carried out hypothesis-driven studies towards synthesising a series of pyrazolo-pyridine carboxylate containing compounds (7a-7m) targeted at reducing deleterious α-Syn aggregation. The target compounds were synthesized through multi-step organic synthesis reactions. From docking studies, compounds 7b, 7g and 7i displayed better interaction with the key residues of α-Syn with values: -6.8, -8.9 and -7.2 Kcal/mol, respectively. In vivo transgenic C. elegans model of Synucleinopathy was used to evaluate the ability of the designed and synthesized compounds to inhibit α-Syn aggregation. These lead compounds 7b, 7g and 7i displayed 1.7, 2.4 and 1.5-fold inhibition of α-Syn with respect to the control. Further, the strategy of employing pyrazolo-pyridine-based compounds worked with success and these scaffolds could be further modified and validated for betterment of endpoints associated with PD.

Inhibition of alpha-synuclein seeded fibril formation and toxicity by herbal medicinal extracts.

BACKGROUND: Recent studies indicated that seeded fibril formation and toxicity of α-synuclein (α-syn) play a main role in the pathogenesis of certain diseases including Parkinson's disease (PD), multiple system atrophy, and dementia with Lewy bodies. Therefore, examination of compounds that abolish the process of seeding is considered a key step towards therapy of several synucleinopathies. METHODS: Using biophysical, biochemical and cell-culture-based assays, assessment of eleven compounds, extracted from Chinese medicinal herbs, was performed in this study for their effect on α-syn fibril formation and toxicity caused by the seeding process. RESULTS: Salvianolic acid B and dihydromyricetin were the two compounds that strongly inhibited the fibril growth and neurotoxicity of α-syn. In an in-vitro cell model, these compounds decreased the insoluble phosphorylated α-syn and aggregation. Also, in primary neuronal cells, these compounds showed a reduction in α-syn aggregates. Both compounds inhibited the seeded fibril growth with dihydromyricetin having the ability to disaggregate preformed α-syn fibrils. In order to investigate the inhibitory mechanisms of these two compounds towards fibril formation, we demonstrated that salvianolic acid B binds predominantly to monomers, while dihydromyricetin binds to oligomeric species and to a lower extent to monomers. Remarkably, these two compounds stabilized the soluble non-toxic oligomers lacking ß-sheet content after subjecting them to proteinase K digestion. CONCLUSIONS: Eleven compounds were tested but only two showed inhibition of α-syn aggregation, seeded fibril formation and toxicity in vitro. These findings highlight an essential beginning for development of new molecules in the field of synucleinopathies treatment.

Immunotherapies in Huntington's disease and α-Synucleinopathies.

Modulation of immune activation using immunotherapy has attracted considerable attention for many years as a potential therapeutic intervention for several inflammation-associated neurodegenerative diseases. However, the efficacy of single-target immunotherapy intervention has shown limited or no efficacy in alleviating disease burden and restoring functional capacity. Marked immune system activation and neuroinflammation are important features and prodromal signs in polyQ repeat disorders and α-synucleinopathies. This review describes the current status and future directions of immunotherapies in proteinopathy-induced neurodegeneration with emphasis on preclinical and clinical efficacies of several anti-inflammatory compounds and antibody-based therapies for the treatment of Huntington's disease and α-synucleinopathies. The review concludes with how disease modification and functional restoration could be achieved by using targeted multimodality therapy to target multiple factors.

Generation of monoclonal antibodies against phosphorylated α-Synuclein at serine 129: Research tools for synucleinopathies.

The majority of α-synuclein (α-syn) within Lewy bodies (LBs) has been reported to be phosphorylated at serine 129 (pS129-α-syn), suggesting a central role for phosphorylation in the pathogenesis of Parkinson's disease (PD) and related synucleinopathies. Various studies have investigated the effect of α-syn phosphorylation but have failed to reach a consensus as to whether this modification accelerates or inhibits α-syn aggregation. Nevertheless, pS129-α-syn is a reliable marker of α-syn aggregates and is widely evaluated in biomarkers and post-mortem studies. While several antibodies specific for pS129-α-syn exist, their reactivity with non-specific antigens appears to be a common challenge. To gain valuable insights into the role of α-syn phosphorylation in disease pathogenesis, antibodies that are highly specific to pS129-α-syn are necessary. In this study, we describe the generation of three mouse monoclonal antibodies (mAbs; 5B9, 6H5 and 9G1) using hybridoma technology. These were thoroughly characterized and validated in combination with our previously generated mAb (PS129), and the commercial ab51253 (Abcam). We demonstrated that our mAbs are highly specific for pS129-α-syn and do not cross react with wild-type α-syn. Results from staining of post-mortem human brain tissue showed that our mAbs detect pS129-α-syn pathology in patients with synucleinopathies. This study highlights three new antibodies as excellent and highly specific research tools to explore the role of pS129-α-syn inclusions in synucleinopathies.

Beta2-Adrenoceptor Agonists in Parkinson's Disease and Other Synucleinopathies.

Evidence supporting the use of ß2AR agonists in synucleinopathies is rapidly growing. Findings come from different scientific approaches. Molecular and immunological data suggest that adrenergic stimulation may decrease both α-synuclein (α-syn) deposition and pro-inflammatory/neurotoxic molecules release. Small open label clinical trials including a total number of 25 Parkinson's disease (PD) patients, in which the ß2AR agonist salbutamol was added to levodopa, suggest a promising symptomatic benefit. In line with these findings, epidemiological studies investigating the risk of PD development suggest that long term exposure to the agonist salbutamol might be protective, while the antagonist propranolol possibly detrimental. Nonetheless, in both lines of investigation the studies performed so far present important limitations. On the clinical side, large randomized controlled trials are lacking, whereas on the epidemiological side the presence of co-morbid conditions (i.e. smoking and essential tremor) potentially influencing PD risk should taken into consideration. In summary, it is our opinion that ß2AR stimulation in synucleinopathies has a rationale and therefore merits further investigation. Graphical Abstract.

GBA1 mutations: Prospects for exosomal biomarkers in α-synuclein pathologies.

The discovery that patients with Gaucher Disease (GD), a rare lysosomal storage disorder, were developing symptoms similar to Parkinson's disease (PD) led to investigation of the relationship between the two seemingly unrelated pathologies. GD, an autosomal recessive disorder, is the result of a biallelic mutation in the gene GBA1, which encodes for the enzyme glucocerebrosidase (GCase). Since the observation of its relation to PD, GBA1 mutations have become recognized as the most common genetic risk factor for development of synucleinopathies such as PD and dementia with Lewy bodies. Although the exact mechanism by which GBA1 mutations promote PD is unknown, current understanding suggests that impaired GCase inhibits lysosomal activity and decreases the overall ability of the cell to degrade proteins, specifically the neuronal protein α-synuclein. Decreased elimination of α-synuclein can lead to its abnormal accumulation and aggregation, an important component of PD development. Further understanding of how decreased GCase activity increases risk for α-synuclein pathology can assist with the development of clinical biomarkers for early detection of synucleinopathies, as well as promote novel treatments tailored for people with a GBA1 mutation. Historically, α-synuclein has not been a reliable biomarker for PD. However, recent research on α-synuclein content within exosomes, which are small vesicles released by cells that carry specific cellular cargo, has yielded encouraging results. Moreover, decreased GCase activity has been shown to influence exosomal contents. Exosomes have emerged as a promising new avenue for the identification of novel biomarkers and therapeutic targets aimed at improving neuronal GCase function and limiting the development of synucleinopathies.

Chronic corticosterone aggravates behavioral and neuronal symptomatology in a mouse model of alpha-synuclein pathology.

Debilitating, yet underinvestigated nonmotor symptoms related to mood/emotion, such as depression, are common in Parkinson's disease. Here, we explore the role of depression and of the amygdala, a brain region robustly linked to mood/emotion, in synucleinopathy. We hypothesized that mood/emotional deficits might accelerate Parkinson's disease-linked symptomatology, including the formation of α-synuclein pathology. We combined elevated corticosterone treatment, modeling chronic stress and depression, with a model of seeded α-synuclein pathology in mouse striatum and assessed behavioral parameters with a focus on mood/emotion, and neuropathology. We report behavioral resilience against α-synuclein proteinopathy in the absence of additional insults, potentially based on hormesis/conditioning mechanisms. Elevated corticosterone, however, reversed α-synuclein pathology-induced behavioral adaptations and was associated with increased dopaminergic cell loss as well as aggravated α-synuclein pathology in specific brain regions, such as the entorhinal cortex. These findings point to elevated glucocorticoids as a risk factor for Parkinson's disease progression and highlight the potential of glucocorticoid level reducing strategies to slow down disease progression in synucleinopathy.

From Yeast to Humans: Leveraging New Approaches in Yeast to Accelerate Discovery of Therapeutic Targets for Synucleinopathies.

Neurodegenerative diseases (ND) represent a growing, global health crisis, one that lacks any disease-modifying therapeutic strategy. This critical need for new therapies must be met with an exhaustive approach to exploit all tools available. A yeast (Saccharomyces cerevisiae) model of α-synuclein toxicity-the protein causally linked to Parkinson's disease and other synucleinopathies-offers a powerful approach that takes advantage of the unique offerings of this system: tractable genetics, robust high-throughput screening strategies, unparalleled data repositories, powerful computational tools, and extensive evolutionary conservation of fundamental biological pathways. These attributes have enabled genetic and small molecule screens that have revealed toxic phenotypes and drug targets that translate directly to patient-derived iPSC neurons. Extending these insights, recent advances in genetic network analyses have generated the first "humanized" α-synuclein network, which has identified druggable proteins and led to validation of the toxic phenotypes in patient-derived cells. Unbiased phenotypic small molecule screens can identify compounds targeting critical proteins within α-synuclein networks. While identification of direct drug targets for phenotypic screen hits represents a bottleneck, high-throughput chemical genetic methods provide a means to uncover cellular targets and pathways for large numbers of compounds in parallel. Taken together, the yeast α-synuclein model and associated tools can reveal insights into underlying cellular pathologies, lead molecules and their cognate targets, and strategies to translate mechanisms of toxicity and cytoprotection into complex neuronal systems.

A small molecule autophagy inducer exerts cytoprotection against α-synuclein toxicity.

α-synucleopathies are protein-misfolding disorders occur primarily due to aggregation and toxicity of α-synuclein. This study characterized the small molecule AGK2 as a modifier of α-synuclein mediated toxicity in an autophagy dependent manner in both yeast and mammalian cell line models. In yeast system, AGK2 enhances autophagy to clear toxic α-synuclein aggregates in an autophagy dependent manner. Autophagy flux analyses revealed that AGK2 induces autophagy especially autolysosomes. Importantly, AGK2 induces autophagy in an mTOR independent manner. These features enable AGK2 to exert cytoprotective potential against α-synuclein mediated toxicity in different model systems.

Animal models of synucleinopathies and how they could impact future drug discovery and delivery efforts.

Introduction: Ample efforts have been carried out to develop efficient disease-modifying drugs of Parkinson's disease and related synucleinopathies. At present, the available animal models appropriate for drug development and delivery for these hitherto incurable disorders are limited. Areas covered: The author, herein, provides their perspectives on classical toxin-based animal models, which have provided some insight into their clinical picture and complex pathogenesis. They also discuss generic and virus-induced models and more recent αSyn transmitter models that reproduce essential clinical and morphological aspects but do not fully replicate the whole spectrum of the human diseases. Yet, these models have, however, provided new insights into the pathogenesis of these disorders. Expert opinion: The recent development of transgenic viral vector-induced αSyn inoculation models and those using induced pluripotent stem cells (iPSCs) in rodents, non-human primates and (rare) primates, reproducing many but not all aspects of the human synucleinopathies and their complex pathogenesis opened the door for the development of successful drugs. Despite these limits, a remarkable amount of work has already been done. However, further attention should be focused on the development of new models to enable better insight into the pathology of these proteinopathies as the basis for the future development of real disease-modifying or even preventive modalities.

Progress in the treatment of Parkinson-Plus syndromes.

Progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB) are the four major proteinopathic neurodegenerative disorders. Currently, there are no disease modifying therapies for these disorders. However, better understanding of the etiopathogenic mechanisms of these disorders has allowed the development of novel therapeutic approaches. These mainly include strategies directed to the pathologic conformational shift, seeding and aggregation, as well as transcellular spread of the proteins that aggregate in the brain which are α-synuclein and tau. Modulation of inflammatory responses and neuroprotection are also targets of interest. A number of clinical trials have been performed and others are ongoing or are planned to address the authentic need for disease modifying treatments. However, challenges exist in terms of accurate early clinical diagnostic criteria and robust outcome measures, and preclinical animal models that would best recapitulate human disease.