Animal models of α-synucleinopathy for Parkinson disease drug development.

BackgroundDementia with Lewy Bodies (DLB) is one of the most common Alzheimer’s Disease (AD)‐related dementias and it is defined by the presence of abnormal cytoplasmic inclusions composed of aggregated α‐synuclein (αsyn) in neuronal soma, known as Lewy bodies (LB). LB often coexists with AD type pathology such as amyloid‐β (Aβ) plaques and neurofibrillary tangles containing hyperphosphorylated tau in several LB dementias, including Parkinson’s Disease Dementia and Lewy Body variant AD. These co‐pathologies likely represent a spectrum of various contributions of shared mechanisms that underlie these diseases. However, the mechanisms of the interaction of these pathological proteins is still poorly understood. Furthermore, there is a lack of mammalian model systems demonstrating robust generation of both Lewy with Aβ and/or tau pathology in the field. Unraveling the mechanistic link between αsyn and AD‐related pathologies might be useful for the treatment of DLB.MethodWe have recently developed a brand‐new transgenic mouse line that expresses A53T αsyn tagged with GFP (A53T Syn‐GFP, AA) that forms LB pathology ∼10x faster than previous models after intracortical/peripheral PFF injection. To investigate the role of tau in αsyn spreading from the periphery to the CNS we crossed the AA mice with the P301S tau transgenic mice (PS19). We have performed monomeric/PPF injection in the gastrocnemius muscle of single and double transgenic mice and carried out a histological analysis of the αsyn and tau pathologies through the spinal cord and the brain.ResultWe found an increase in the number of αsyn+ inclusion induced by PFF injection in the spinal cord and the brain of PS19/AA mice compared to single AA mice. Interestingly, we found sex differences in αsyn pathology being higher in females than males. Furthermore, monomeric αsyn caused reduced but some αsyn inclusions in the spinal cord after muscle injection.ConclusionOur preliminary data shows an increase in αsyn pathology and spreading from the periphery to the brain in the presence of tau pathology suggesting a link between αsyn and tau pathologies that could represent potential treatment target for the treatment of DLB patients.

BackgroundAbnormal folding, aggregation and spreading of alpha-synuclein (αsyn) is a mechanistic hypothesis for the progressive neuropathology in Parkinson's disease (PD). Spread of αsyn between cells is supported by clinical, neuropathological and experimental evidence. It has been proposed that a pro-inflammatory micro-environment in response to αsyn can promote its aggregation. We have previously shown that allelic differences in the major histocompatibility complex class two transactivator (Mhc2ta) gene, located in the VRA4 locus, alter MHCII expression levels, microglial activation and antigen presentation capacity in rats upon human αsyn over-expression. In addition, Mhc2ta regulated dopaminergic neurodegeneration and the extent of motor impairment. The purpose of this study was to determine whether Mhc2ta regulates αsyn aggregation, propagation and dopaminergic pathology in an αsyn pre-formed fibril (PFF)-seeded in vivo model of PD. MethodsThe DA and DA.VRA4 congenic rat strains share background genome but display differential microglial antigen presenting capacity due to different Mhc2ta alleles in the VRA4 locus. PFFs of human αsyn or BSA solution were injected unilaterally to the striatum of DA and DA.VRA4 rats two weeks after ipsilateral administration of recombinant adeno-associated virus (rAAV) vectors carrying human αsyn or GFP to the substantia nigra pars compacta. Behavioural assessment was performed at 2, 5 and 8 weeks while histological evaluation of αsyn pathology, inflammation and neurodegeneration as well as determination of serum cytokine profiles were performed at 8 weeks. ResultsrAAV-mediated expression of human αsyn in nigral dopaminergic neurons combined with striatal PFF administration induced enhanced αsyn pathology in DA.VRA4 compared to DA rats. Mhc2ta thus significantly regulated the seeding, propagation and toxicity of αsyn in vivo. This was reflected in terms of wider extent and anatomical distribution of αsyn inclusions, ranging from striatum to the forebrain, midbrain, hindbrain and cerebellum in DA.VRA4. Compared to DA rats, DA.VRA4 also displayed enhanced motor impairment and dopaminergic neurodegeneration as well as higher levels of the proinflammatory cytokines IL-2 and TNFα in serum. ConclusionsWe conclude that the key regulator of MHCII expression, Mhc2ta, modulates neuroinflammation, αsyn-seeded Lewy-like pathology, dopaminergic neurodegeneration and motor impairment. This makes Mhc2ta and microglial antigen presentation promising therapeutic targets for reducing the progressive neuropathology and clinical manifestations in PD.

Parkinson's disease (PD) presents with both motor and non-motor symptoms, including depression and anxiety, which often precede motor onset, yet the mechanisms linking these affective symptoms to PD pathology remain unclear. In this study, we investigated the bidirectional relationship between depression/anxiety behaviors and α-synuclein (α-syn) propagation using A53T α-syn transgenic mice subjected to chronic restraint stress (CRS) and/or intrastriatal injection of α-syn preformed fibrils (PFFs). Behavioral testing and immunohistochemical analyses revealed that CRS enhanced PFF-induced α-syn propagation and exacerbated depression/anxiety-like behaviors, while α-syn propagation was associated with aggravated CRS-induced behavioral deficits, indicating a potential reciprocal association that could contribute to accelerating PD progression. This interaction was mediated by the neuronal activity marker c-Fos. Pharmacological inhibition of c-Fos with T5224 mitigated both behavioral and pathological changes, and mGluR5 activation was found to partially contribute to c-Fos induction and α-syn spread. Together, these findings highlight a feedback interaction between affective symptoms and α-syn pathology in PD, mediated in part by neuronal activity-dependent mechanisms involving c-Fos and mGluR5, and suggest that early interventions targeting both neuronal activity and α-syn propagation may slow PD progression and improve patient quality of life.

Braak's theory described Parkinson's disease (PD) progression as prion-like α-synuclein (αSyn) spreading, which fundamentally subverts the understanding of pathogenesis. The pathological αSyn spreading pathway includes uptake, propagation, and release. However, the previous disease models were limitedly focusing on amyloid propagation/aggregation, which significantly impedes the mechanism exploration in spreading pathways and related therapeutic development. The spreading model can be achieved using recombinant αSyn preformed fibrils (PFFs), which seeds endogenous αSyn monomer to aggregation and causes substantial pathology and neurotoxicity. Here, we determined that dihydromyricetin (DHM), a natural flavonoid extracted from Ampelopsis grossedentata, can promote the fibrillization of prion-like PFF and induce propagation to form a distinct strain. Furthermore, administration of DHM significantly reduced prion-like PFF-induced propagation and neurotoxicity. The discovery of inducing infectious and neurotoxic PFF to a nontoxic strain resulting in neuron protection via promoting the fibrillization of PFF rather than inhibiting advances the understanding of the prion-like spreading mechanism and helps in developing treatments against PD and related α-synucleinopathies.

Protein aggregation and accumulation are common pathological hallmarks in neurodegenerative diseases. To efficiently clear and eliminate such aggregation becomes an important cellular strategy for cell survival. Lewy bodies inclusion and aggregation of α-Synuclein (α-Syn) during the pathogenesis of Parkinson’s disease (PD) serve as a good example and are potentially linked to other pathological PD features such as progressive dopaminergic neuron cell death, behavioral defects, and nonmotor symptoms like anosmia, cognitive impairment, and depression. Years of research have revealed a variety of mechanisms underlying α-Syn aggregation, clearance, and spread. Particularly, vesicular routes associated with the trafficking of α-Syn, leading to its aggregation and accumulation, have been shown to play vital roles in PD pathogenesis. How α-Syn proteins propagate among cells in a prion-like manner, either from or to neurons and glia, via means of uptake or secretion, are questions under active investigation and have been of central interest in the field of PD study. This review covers components and pathways of possible vesicular routes involved in α-Syn trafficking. Events including but not limited to exocytosis and endocytosis will be discussed within the context of an overall cellular trafficking theme. Recent advances on α-Syn trafficking mechanisms and their significance in mediating PD pathogenesis will be thoroughly reviewed, ending with a discussion on the advantages and limitations of different animal PD models.

BackgroundPathological α-synuclein (α-syn) can spread from the gut to the central nervous system (CNS), with CD4 + T cells playing a key role in this process. Lymphocyte activation gene 3 (LAG3) is involved in intestinal inflammation, regulates CD4 + T cell proliferation and function, and can specifically bind to pathological α-syn during cell-to-cell transmission. However, it remains unclear whether LAG3 is involved in the spread of pathological α-syn from the gut to the brain.MethodsWe utilized LAG3 knockout mice, combined with injection of α-syn preformed fibril (PFF) into the longitudinal and intermediate muscle layers of the pylorus and duodenum to model Parkinson’s disease (PD). We used Immunohistochemistry staining, Western Blot, Flow cytometry to detect the changes of TH, α-syn, pro-inflammatory factors, barrier-related proteins and CD4 + T cells differentiation.ResultsOur results show that LAG3 knockout partially alleviates psychological and behavioral deficits, dopamine system damage, and the gut-to-brain transmission of α-syn, which correlates with enhanced regulatory T cell (Treg) cell proliferation. Furthermore, LAG3 knockout improved intestinal dysfunction and increased the expression of tight junction proteins in both the gut and the blood-brain barrier (BBB). In CD4 + T cells isolated from the spleen, LAG3 knockout suppressed the aggregation of α-syn PFF, thereby inhibiting the toxic T-cell response induced by α-syn PFF. LAG3 deficiency also enhanced the IL-2/STAT5 signaling pathway, which regulates Treg proportions both in vivo and in vitro.ConclusionsOur findings demonstrated that LAG3 intrinsically limits Treg cell proliferation and function in the environment with pathological α-syn and promotes the gut-to-brain transmission of α-syn.Graphical abstractLAG3 deletion delays α-syn gut-to-brain transmission. Compared to wild-type mice, the proliferation of Treg is dramatically increased with LAG3 knockout, resulting in delayed pathological α-syn gut-to-brain transmission and toxicity.

Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) are characterized by the aberrant accumulation of intracytoplasmic misfolded and aggregated α-synuclein (α-Syn), resulting in neurodegeneration associated with inflammation. The propagation of α-Syn aggregates from cell to cell is implicated in the spreading of pathological α-Syn in the brain and disease progression. We and others demonstrated that antibodies generated after active and passive vaccinations could inhibit the propagation of pathological α-Syn in the extracellular space and prevent/inhibit disease/s in the relevant animal models. We recently tested the immunogenicity and efficacy of four DNA vaccines on the basis of the universal MultiTEP platform technology in the DLB/PD mouse model. The antibodies generated by these vaccines efficiently reduced/inhibited the accumulation of pathological α-Syn in the different brain regions and improved the motor deficit of immunized female mice. The most immunogenic and preclinically effective vaccine, PV-1950D, targeting three B-cell epitopes of pathological α-Syn simultaneously, has been selected for future IND-enabling studies. However, to ensure therapeutically potent concentrations of α-Syn antibodies in the periphery of the vaccinated elderly, we developed a recombinant protein-based MultiTEP vaccine, PV-1950R/A, and tested its immunogenicity in young and aged D-line mice. Antibody responses induced by immunizations with the PV-1950R/A vaccine and its homologous DNA counterpart, PV-1950D, in a mouse model of PD/DLB have been compared.

Inflammation-associated peripheral blood cells and serum lipid levels are associated with Parkinson's disease.

Olfactory proteotyping: towards the enlightenment of the neurodegeneration

Amyloid-Beta (Aβ) Plaques Promote Seeding and Spreading of Alpha-Synuclein and Tau in a Mouse Model of Lewy Body Disorders with Aβ Pathology

BackgroundDysregulated leukotriene signaling is proposed to be involved in pathogenesis of Parkinson's disease (PD).ObjectiveThe objective was to examine the safety and tolerability of montelukast, a cysteinyl‐leukotriene receptor1 and GPR17 antagonist, in patients with PD. Secondary outcomes were target engagement, effects on PD signs/symptoms, and central neuroinflammation.MethodsFifteen PD patients were recruited to a 12‐week open‐label trial of 20 mg bi‐daily montelukast treatment. Patients underwent ratings with the Movement Disorder Society Unified Parkinson Disease Rating Scale (MDS‐UPDRS), the Montreal Cognitive Assessment (MoCA), Beck's Depression Inventory (BDI), Parkinson's Disease Questionnaire‐39 (PDQ‐39), [11C]PBR28‐PET, and lumbar punctures before and during montelukast treatment.ResultsAll patients completed the study. Three patients reported loose stool. No serious adverse events related to treatment were reported. MDS‐UPDRS‐Total scores improved by 6.9 points. Very low levels of montelukast were detected in all cerebrospinal fluid (CSF) samples and resulted in a reduction in inflammation/metabolism markers. [11C]PBR28 binding was lowered in high, but not mixed, affinity binders after montelukast.ConclusionsMontelukast crosses the blood–brain barrier at very low levels and is well tolerated and safe in PD patients. Preliminary effects on neuroinflammation and clinical scores motivate a future randomized controlled trial (RCT) in PD. © 2025 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Leucine-rich repeat kinase 2 is a potential therapeutic target for the treatment of Parkinson's disease, and clinical trials of leucine-rich repeat kinase 2 inhibitors are in development. The objective of this study was to evaluate phosphorylation of a new leucine-rich repeat kinase 2 substrate, Rab10, for potential use as a target engagement biomarker and/or patient enrichment biomarker for leucine-rich repeat kinase 2 inhibitor clinical trials. Peripheral blood mononuclear cells and neutrophils were isolated from Parkinson's disease patients and matched controls, and treated ex vivo with a leucine-rich repeat kinase 2 inhibitor. Immunoblotting was used to measure levels of leucine-rich repeat kinase 2 and Rab10 and their phosphorylation. Plasma inflammatory cytokines were measured by multiplex enzyme-linked immunosorbent assay. Mononuclear cells and neutrophils of both controls and Parkinson's disease patients responded the same to leucine-rich repeat kinase 2 inhibitor treatment. Leucine-rich repeat kinase 2 levels in mononuclear cells were the same in controls and Parkinson's disease patients, whereas leucine-rich repeat kinase 2 was significantly increased in Parkinson's disease neutrophils. Rab10 T73 phosphorylation levels were similar in controls and Parkinson's disease patients and did not correlate with leucine-rich repeat kinase 2 levels. Immune-cell levels of leucine-rich repeat kinase 2 and Rab10 T73 phosphorylation were associated with plasma inflammatory cytokine levels. Rab10 T73 phosphorylation appears to be a valid target engagement biomarker for potential use in leucine-rich repeat kinase 2 inhibitor clinical trials. However, a lack of association between leucine-rich repeat kinase 2 and Rab10 phosphorylation complicates the potential use of Rab10 phosphorylation as a patient enrichment biomarker. Although replication is required, increased leucine-rich repeat kinase 2 levels in neutrophils from Parkinson's disease patients may have the potential for patient stratification. leucine-rich repeat kinase 2 activity in peripheral immune cells may contribute to an inflammatory phenotype. © 2018 International Parkinson and Movement Disorder Society.

Small Molecule Inhibitors for Precise Inhibition of Alpha-Synuclein Oligomer Generation in Parkinson’s Disease

Genetic variation in the leucine-rich repeat kinase 2 (LRRK2) gene is associated with risk of familial and sporadic Parkinson’s disease (PD). To support clinical development of LRRK2 inhibitors as disease-modifying treatment in PD biomarkers for kinase activity, target engagement and kinase inhibition are prerequisite tools. In a combined proteomics and phosphoproteomics study on human peripheral mononuclear blood cells (PBMCs) treated with the LRRK2 inhibitor Lu AF58786 a number of putative biomarkers were identified. Among the phospho-site hits were known LRRK2 sites as well as two phospho-sites on human Rab10 and Rab12. LRRK2 dependent phosphorylation of human Rab10 and human Rab12 at positions Thr73 and Ser106, respectively, was confirmed in HEK293 and, more importantly, Rab10-pThr73 inhibition was validated in immune stimulated human PBMCs using two distinct LRRK2 inhibitors. In addition, in non-stimulated human PBMCs acute inhibition of LRRK2 with two distinct LRRK2 inhibitor compounds reduced Rab10-Thr73 phosphorylation in a concentration-dependent manner with apparent IC50’s equivalent to IC50’s on LRRK2-pSer935. The identification of Rab10 phosphorylated at Thr73 as a LRRK2 inhibition marker in human PBMCs strongly support inclusion of assays quantifying Rab10-pThr73 levels in upcoming clinical trials evaluating LRRK2 kinase inhibition as a disease-modifying treatment principle in PD.

Background:The death of dopaminergic neurons in Parkinson's disease (PD) appears to have various causes, including oxidative stress, excitotoxicity, mitochondrial dysfunction (and associated apoptosis), ubiquitin/proteasomal dysfunction, and inflammation, any of which could in principle be the therapeutic target of a neuroprotective drug. The biology of dopaminergic neurons offers further potential targets, involving neurotrophic factors, dopamine-neuron genes, and even neurogenesis.Objective:To outline each hypothetical neuroprotective mechanism, the evidence suggesting its relevance to PD, and the research on pharmacologic intervention.Methods:A PubMed search was conducted to identify relevant preclinical and clinical literature published between 1989 and 2009. Additional articles were identified by reviewing the reference lists of papers selected in the original search. To circumscribe the survey and facilitate consideration of the conditions required for a neuroprotective effect, emphasis was placed on a single drug class, dopamine agonists, and in particular pramipexole.Review of the field:In a variety of in vitro and in vivo PD models, pramipexole exhibited preclinical evidence of neuroprotective actions of all hypothesized types, and in human neuroimaging studies it slowed the rate of loss of markers of dopaminergic function, consistent with drug-conferred neuroprotection in PD itself. Interpretation of the preclinical data was hampered by differences among models and by uncertainties concerning each model's mimicry of PD. Overall, the identified neuroprotection almost always required pretreatment (i.e., before insult) and high drug concentration. Interpretation of the clinical data was hampered by absence of placebo control and of a direct measure of neuroprotection.Conclusions:Although the evidence is promising, neuroprotection in PD remains an elusive goal. In whatever form it emerges, neuroprotective therapy would be a strong argument against deferring PD treatment until symptoms are a significant life impediment, and thus would add urgency to early PD identification.