α-synuclein monoclonal antibodies in Parkinson's disease: A failed promise or unmet potential?

2021 Workshop: Neurodegenerative Diseases in the Gut-Brain Axis—Parkinson's Disease

The Discovery of α-Synuclein in Lewy Pathology of Parkinson's Disease: The Inspiration of a Revolution.

Cognitive impairment is a common non-motor symptom of Parkinson’s disease (PD) and a defining feature of Dementia with Lewy Bodies (DLB). Although many cognitive domains can be affected, impairments in visuospatial/perceptual function are relatively specific for PD and DLB compared to other dementias. Across populations, cognitive impairments correlate with the presence of α-synuclein (α-syn) pathology in limbic and neocortical brain regions. However, the specific role that α-syn pathology plays in driving cortical circuit dysfunction and cognitive impairment remains controversial. We hypothesized that inducing α-syn pathology in visual cortex in mice would impair neuronal activity and encoding of visual information, leading to visuoperceptual impairments. To test this hypothesis, we injected α-syn pre-formed fibrils (PFF) into primary visual cortex (V1) to seed endogenous α-syn pathology. Using longitudinal in vivo two-photon (2P) calcium imaging over 6 months, we recorded visually evoked activity of pyramidal cells in layer 2/3 (L2/3) and quantified α-syn pathology using C05–05, a fluorescent ligand that binds aggregated α-syn. Injection of PFFs led to the formation of sparse Lewy-like pathology in V1 and other anatomically connected regions. Measuring population activity, we found a greater percentage of neurons in PFF-injected mice were responsive to visual stimuli with lower direction selectivity compared to controls at 4–5 months post-injection (MPI). Within PFF-injected mice, neurons with large somatic Lewy-like inclusions had significantly lower visually evoked activity compared to neighboring neurons without inclusions. Conversely, the activity of neurons without somatic inclusions showed increased activity, positively correlated with the nearby burden of α-syn pathology. Measuring visuoperceptual function using a head-fixed coherent motion discrimination task, we found no impairments in visuoperceptual ability in PFF-injected mice up to 6 MPI. Our results demonstrate, for the first time in vivo, that α-syn pathology leads to cell autonomous reductions in neuronal activity and reciprocal changes in local population activity that may be compensatory, helping to preserve visuoperceptual function. Reflecting the early stages of neocortical α-syn pathology, our model provides a framework for future studies incorporating risk factors for dementia in PD to better understand the heterogeneity of cognitive symptoms and α-syn pathology across patients.

Alpha-synucleinopathies are neurodegenerative diseases characterized by the spread of alpha-synuclein (α-syn) aggregates throughout the central nervous system in a stereotypical manner. These diseases include Lewy body disease (LBD), which encompass Dementia with Lewy bodies (DLB), Parkinson’s Disease (PD), and Parkinson’s Disease Dementia (PDD), and Multiple System Atrophy (MSA). LBD and MSA chiefly contain α-syn aggregates in neurons and oligodendrocytes, respectively, although glial α-syn pathology in LBD is increasingly being recognized. Semi-quantitative and machine learning-based quantifications of neuronal, oligodendrocytic and astrocytic α-syn pathology were implemented on a cohort of LBD and MSA post-mortem tissue samples. The neuroanatomical distribution of each cell-type specific α-syn pathology was evaluated using conditional probability matrices and Subtype and Stage Inference (SuStaIn) algorithm. We revealed extensive glial α-syn pathology in LBD, emphasizing the disease- and region-specific profile of astrocytic α-syn pathology, which was absent in MSA and minimal in the substantia nigra of LBD. Furthermore, we have described distinct morphologies of astrocytic α-syn pathology, which were found to correlate with the density of astrocytic α-syn inclusions. Astrocytic α-syn pathology was mainly centered in the amygdala and exhibited a unique stereotypical progression whilst oligodendrocytes displayed a distribution akin to the established neuronal progression pattern. SuStaIn modeling was further used to test for heterogeneity in the spatiotemporal progression, revealing that a subset of cases might follow an alternative pattern. Based on these findings, we introduce a novel multimodal progression framework that integrates, for the first time, the temporal and spatial progression of astrocytic and oligodendrocytic α-syn pathology alongside neuronal pathology in PD, providing further information regarding the role of neurons and glia in disease pathogenesis.

SUMMARYα-synuclein (α-syn) aggregation and accumulation drive neurodegeneration in Parkinson’s disease (PD). The substantia nigra of patients with PD contains excess iron, yet the underlying mechanism accounting for this iron accumulation is unclear. Here, we show that misfolded α-syn activates microglia, which release interleukin 6 (IL-6). IL-6, via its trans-signaling pathway, induces changes in the neuronal iron transcriptome that promote ferrous iron uptake and decrease cellular iron export via a pathway we term the cellular iron sequestration response, or CISR. The brains of patients with PD exhibit molecular signatures of the IL-6-mediated CISR. Genetic deletion of IL-6, or treatment with the iron chelator deferiprone, reduces pathological α-syn toxicity in a mouse model of sporadic PD. These data suggest that IL-6-induced CISR leads to toxic neuronal iron accumulation, contributing to synuclein-induced neurodegeneration.

Progressive aggregation of the protein alpha-synuclein (α-syn) and loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) are key histopathological hallmarks of Parkinson’s disease (PD). Accruing evidence suggests that α-syn pathology can propagate through neuronal circuits in the brain, contributing to the progressive nature of the disease. Thus, it is therapeutically pertinent to identify modifiers of α-syn transmission and aggregation as potential targets to slow down disease progression. A growing number of genetic mutations and risk factors has been identified in studies of familial and sporadic forms of PD. However, how these genes affect α-syn aggregation and pathological transmission, and whether they can be targeted for therapeutic interventions, remains unclear. We performed a targeted genetic screen of risk genes associated with PD and parkinsonism for modifiers of α-syn aggregation, using an α-syn preformed-fibril (PFF) induction assay. We found that decreased expression of Lrrk2 and Gba modulated α-syn aggregation in mouse primary neurons. Conversely, α-syn aggregation increased in primary neurons from mice expressing the PD-linked LRRK2 G2019S mutation. In vivo, using LRRK2 G2019S transgenic mice, we observed acceleration of α-syn aggregation and degeneration of dopaminergic neurons in the SNpc, exacerbated degeneration-associated neuroinflammation and behavioral deficits. To validate our findings in a human context, we established a novel human α-syn transmission model using induced pluripotent stem cell (iPS)-derived neurons (iNs), where human α-syn PFFs triggered aggregation of endogenous α-syn in a time-dependent manner. In PD subject-derived iNs, the G2019S mutation enhanced α-syn aggregation, whereas loss of LRRK2 decreased aggregation. Collectively, these findings establish a strong interaction between the PD risk gene LRRK2 and α-syn transmission across mouse and human models. Since clinical trials of LRRK2 inhibitors in PD are currently underway, our findings raise the possibility that these may be effective in PD broadly, beyond cases caused by LRRK2 mutations.

Dysregulation of microRNA gene expression has been implicated in many neurodegenerative diseases, including Parkinson's disease. However, the individual dysregulated microRNAs remain largely unknown. Previous meta-analyses have highlighted several microRNAs being differentially expressed in post-mortem Parkinson's disease and Alzheimer's disease brains versus controls, but they were based on small sample sizes. In this study, we quantified the expression of the most compelling Parkinson's and Alzheimer's disease microRNAs from these meta-analyses ('candidate miRNAs') in one of the largest Parkinson's/Alzheimer's disease case-control post-mortem brain collections available (n = 451), thereby quadruplicating previously investigated sample sizes. Parkinson's disease candidate microRNA hsa-miR-132-3p was differentially expressed in our Parkinson's (P = 4.89E-06) and Alzheimer's disease samples (P = 3.20E-24) compared with controls. Alzheimer's disease candidate microRNAs hsa-miR-132-5p (P = 4.52E-06) and hsa-miR-129-5p (P = 0.0379) were differentially expressed in our Parkinson's disease samples. Combining these novel data with previously published data substantially improved the statistical support (α = 3.85E-03) of the corresponding meta-analyses, clearly implicating these microRNAs in both Parkinson's and Alzheimer's disease. Furthermore, hsa-miR-132-3p/-5p (but not hsa-miR-129-5p) showed association with α-synuclein neuropathological Braak staging (P = 3.51E-03/P = 0.0117), suggesting that hsa-miR-132-3p/-5p play a role in α-synuclein aggregation beyond the early disease phase. Our study represents the largest independent assessment of recently highlighted candidate microRNAs in Parkinson's and Alzheimer's disease brains, to date. Our results implicate hsa-miR-132-3p/-5p and hsa-miR-129-5p to be differentially expressed in both Parkinson's and Alzheimer's disease, pinpointing shared pathogenic mechanisms across these neurodegenerative diseases. Intriguingly, based on publicly available high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation data, hsa-miR-132 may interact with SNCA messenger RNA in the human brain, possibly pinpointing novel therapeutic approaches in fighting Parkinson's disease.

Convergence of Heat Shock Protein 90 with Ubiquitin in Filamentous α-Synuclein Inclusions of α-Synucleinopathies

α-Synuclein (α-Syn) pathology is present in 30-50 % of Alzheimer's disease (AD) patients, and its interactions with tau proteins may further exacerbate pathological changes in AD. However, the specific role of different aggregation forms of α-Syn in the progression of AD remains unclear. To explore the relationship between various aggregation types of CSF α-Syn and Alzheimer's disease progression. We conducted a retrospective analysis of data from the Alzheimer's Disease Neuroimaging Initiative (ADNI) to examine the association between different α-Syn aggregation forms-Syn0 (no detectable α-Syn aggregates) and Syn1 (α-Syn aggregates detected, resembling those found in Parkinson's disease)-with the pathological and clinical features of AD. Additionally, we evaluated their potential as predictors of conversion from mild cognitive impairment (MCI) to AD. The ADNI database. A total of 250 participants, including 70 cognitively normal controls, 119 patients diagnosed with MCI, and 61 patients diagnosed with AD. Pearson correlation was employed to assess the relationship between α-Syn levels and cerebrospinal fluid (CSF) biomarkers, including total tau (T-tau), phosphorylated tau (p-tau), and amyloid-β42 (Aβ42). Multivariate Cox proportional hazards models were applied, adjusting for APOE4 status, age, and sex, to determine the association between α-Syn forms and AD-related pathological and clinical outcomes. Kaplan-Meier curves were used to evaluate the prognostic value of different α-Syn aggregation states in predicting the conversion from MCI to AD. Compared with controls, overall MCI and AD patients had elevated α-Syn levels. Notably, in the α-Syn0 group, α-Syn levels were increased in the MCI patients and further increased in AD patients, whereas in the α-Syn1 group, α-Syn levels did not significantly differ across diagnostic groups. Both in the α-Syn0 and α-Syn1 groups, α-Syn levels were found to correlate more strongly with CSF tau levels than with Aβ42, indicating a possible role for α-Syn in tau-related pathology in AD. Importantly, α-Syn0-AD patients exhibited more rapid cognitive decline and greater hippocampal atrophy than α-Syn1-AD patients. However, MCI patients with CSF α-Syn1 aggregation status had an increased risk of conversion to AD. CSF α-Syn is associated with tau pathology and neurodegeneration in Alzheimer's disease. The distinct aggregation profiles of α-Syn serve as valuable biomarkers, offering insights into differing prognoses in AD and aiding in the prediction of early disease progression.

Development and characterization of monoclonal antibodies for Parkinson’s Diseases and Related Disorders Najlaa Al-Thani1, Nour Majbour2, Muneera Fayyad3, N. Vaikath2, and Omar M. A. El-Agnaf2,3 1 Carnegie Mellon University in Qatar 2Neurological Disorders Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation, PO Box 5825, Doha, Qatar. 3Life Sciences Division, College of Science and Engineering, Hamad Bin Khalifa University (HBKU), Education City, Qatar Foundation, PO Box 5825, Doha, Qatar Neurodegenerative diseases are the leading cause for disability in the world and one of the biggest burdens on our societies. Parkinson's Disease (PD) is the second most common neurodegenerative disease after Alzheimer's Disease (AD). PD is a chronic, progressive and irreversible disorder. The social and economic burden imposed by PD is significantly increasing as populations age. PD was first described by James Parkinson, an English physician, in his assay “Shaking Palsy” over 100 years ago. Although in less than a year PD will enter its next century, many aspects of the disease remain to be elucidated. PD can be sporadic or inherited, and in both cases it is accompanied by degeneration of dopaminergic neurons in the SN. Lewy bodies (LBs) and Lewy nurites (LNs) are the main pathological features of PD, where alpha-synuclein (α-syn) is the main component. α-Syn is a pre-synaptic neuronal protein and its aggregation and dysfunction is linked to a number of neurodegenerative disorders named as “synucleinopathies”. Synucleinopathies mainly refer to PD, dementia with Lewy bodies and multiple system atrophy. α-Syn is 140 amino-acid protein that is highly abundant in the brain and can also be found in red blood cells, plasma, cerebrospinal fluid (CSF) and saliva. The protein is natively unfolded, however, accumulating evidence indicate that enhanced oligomerization and aggregation of α-syn is associated with increased toxicity. Extensive efforts have been put into the elucidation of the mechanisms responsible for the polymerization and aggregation of α-syn. α-Syn undergoes several post-translational modifications such as phosphorylation, truncation or ubiquitination. A better understanding of the role of α-syn post-translational modifications will help to elucidate the exact role of α-syn in the pathogenesis of PD, paving the way to develop new diagnostic and therapeutic strategies for synucleinopathies. Truncated α-syn was found to be abundant in the brains of PD and DLB patients, suggesting that truncated α-syn may play a normal physiological role as well as a pathological one. C-terminal truncated α-syn as it exhibited a higher propensity to fibrilize in comparison with WT full length α-syn. In vitro studies that truncated α-syn specifically C-terminal residues 109-140 promoted aggregation presumably through nucleation formation. Antibodies that recognize both full length and c-terminally truncated a-synuclein are available, however, to study the role of c-terminally truncated a-synuclein in PD, specific antibodies are needed. In this project, we have generated and thoroughly characterized monoclonal antibodies against c-terminally truncated α-syn at Asn122. Following the selection of the stable clones, only the clones of IgG antibody were selected. All the clones were passaged multiple times to identify stable clones and subjected to single cell cloning to achieve monoclonality. The selected clones were cultured on a larger scale, mass culture, and the culture supernatant was then purified using Protein G affinity purification. The mAbs were highly selective for 122 α-syn, and didn't cross-react with 140 α-syn. The specificity and sensitivity of these antibodies were assessed by an array of biochemical methods including slot-blotting, western blotting, ELISA and immunohistochemical analysis. The potential of our antibodies can be explored as diagnostic or therapeutic tools for PD and related disorders.

In vivo functional and structural brain imaging of synucleinopathies in humans have provided a rich new understanding of the affected networks across the cortex and subcortex. Despite this progress, the temporal relationship between α-synuclein (α-syn) pathology and the functional and structural changes occurring in the brain is not well understood. Here, we examine the temporal relationship between locomotor ability, brain microstructure, functional brain activity, and α-syn pathology by longitudinally conducting rotarod, diffusion magnetic resonance imaging (MRI), resting-state functional MRI (fMRI), and sensory-evoked fMRI on 20 mice injected with α-syn fibrils and 20 PBS-injected mice at three timepoints (10 males and 10 females per group). Intramuscular injection of α-syn fibrils in the hindlimb of M83+/- mice leads to progressive α-syn pathology along the spinal cord, brainstem, and midbrain by 16 weeks post-injection. Our results suggest that peripheral injection of α-syn has acute systemic effects on the central nervous system such that structural and resting-state functional activity changes occur in the brain by four weeks post-injection, well before α-syn pathology reaches the brain. At 12 weeks post-injection, a separate and distinct pattern of structural and sensory-evoked functional brain activity changes was observed that are co-localized with previously reported regions of α-syn pathology and immune activation. Microstructural changes in the pons at 12 weeks post-injection were found to predict survival time and preceded measurable locomotor deficits. This study provides preliminary evidence for diffusion and fMRI markers linked to the progression of synuclein pathology and has translational importance for understanding synucleinopathies in humans.SIGNIFICANCE STATEMENT α-Synuclein (α-syn) pathology plays a critical role in neurodegenerative diseases such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. The longitudinal effects of α-syn pathology on locomotion, brain microstructure, and functional brain activity are not well understood. Using high field imaging, we show preliminary evidence that peripheral injection of α-syn fibrils induces unique patterns of functional and structural changes that occur at different temporal stages of α-syn pathology progression. Our results challenge existing assumptions that α-syn pathology must precede changes in brain structure and function. Additionally, we show preliminary evidence that diffusion and functional magnetic resonance imaging (fMRI) are capable of resolving such changes and thus should be explored further as markers of disease progression.

Occupational exposures to magnetic fields and neurodegenerative disease risks

Alpha-synuclein (a-syn) has been implicated in the etiopathogenesis of Parkinson’s disease (PD) based on the observation that mutations in the protein cause a familial form of the disease. That duplication and triplication of the wild type gene can also lead to a familial form of PD suggests that increased expression of the normal protein is itself sufficient to cause the disease. The observation that Lewy pathology is found in formerly healthy young dopamine neurons that have been implanted into the striatum of PD patients has raised the possibility that a-syn is a prion and that PD is a prion disorder. In support of this concept, it has now been demonstrated that a-syn can be taken up dopamine neurons in both transgenic and wild type rodents leading to inclusion body formation, neurodegeneration, behavioral abnormalities and transfer/ transport to neighboring regions with extension of the disease process (reviewed in reference 2). In PD, there has been considerable interest in the possibility that this sequence might begin in the periphery and specifically in the GI system where it is postulated that a-syn pathology might initiate and then spread to involve brain stem structures. Confirmation of this concept would greatly enhance our understanding of the nature of the PD pathologic process, provide novel targets for a neuroprotective therapy and offer the opportunity to identify a biomarker for diagnosing patients at an early or prodromal stage of the disease. Indeed, it may be necessary to introduce a neuroprotective strategy at an early stage as recent studies suggest that dopamine neuronal degeneration is largely complete within a few years of diagnosis based on the traditional motor features. While a-syn pathology in the SNc is widely used as the seminal post-mortem diagnostic marker for confirmation of the clinical diagnosis of PD, it is now wellappreciated that a-syn pathology is also present within multiple structures in the brain, spinal cord and peripheral autonomic nervous system of PD patients, and specifically within the GI system. Indeed, a-syn pathology has been reported on colonic biopsies from asymptomatic individuals who years later went on to develop PD. This supports the Braak hypothesis which proposes that a-syn pathology spreads in a predictable and sequential manner, affecting the dorsal motor nucleus (DMN) of the vagus nerve at an early stage of the disease and only involving the SNc in a mid-stage of the illness. Terminals of the dorsal motor nucleus reside in the submucosal layer of the GI tract, mere millimeters from the lumen. This raises the possibility that exposure to a toxin or infectious agent could initiate a-syn accumulation and aggregation in the periphery with subsequent spread to the central nervous system (CNS.). These observations are consistent with epidemiologic studies indicating that constipation is a risk factor for PD, and may in fact represent a prodromal phase of the disorder. Accordingly there has been great interest in determining if asyn pathology can be detected in the GI system prior to the onset of the classic motor features of PD. Several articles published in the current and recent issues of Movement Disorders provide information relevant to the concept that pathologic a-syn might initially be expressed in the GI system, and may serve as a nidus for transmission to the DMN and other brain stem and cerebral structures. Confirmation of this hypothesis would have great importance for defining a potential biomarker of the disease for early diagnosis and for better understanding the nature of the origin of PD pathology.

Stress granules (SGs) are membraneless organelles that are constituted primarily of untranslated messenger ribonucleoproteins, whose assembly occurs via liquid-liquid phase separation (LLPS) in response to various cellular stressors and that play a critical role in maintaining proteostasis. Increasing evidence indicates that chronic stress can disrupt the tightly regulated dynamics of SGs assembly and disassembly, leading to their persistence. These pathological SGs potentially serve as nucleation sites for aberrant protein aggregation in several neurodegenerative disorders (NDDs)2. Focusing on Parkinson’s disease (PD), current knowledge on the dysregulation of SGs assembly and disassembly in relation to α-Synuclein (α-Syn) aggregation is only based on cellular models, with no evidence from human brain tissue. It is well established that, under specific conditions, α-Syn can form condensates regulated by microtubule dynamics, promoting amyloid fibril formation via LLPS, and that α-Syn interacts with several RNA-binding proteins (RBPs) involved in SGs formation. Based on this we aim to determine whether: i) α-Syn pathology and SGs are associated in post-mortem human brains obtained from PD patients and ii) α-Syn aggregates within SGs under conditions of acute and chronic stress in a neuronal cell model (SH-SY5Y cells) and whether this process affects SGs homeostasis. Immunohistochemical analysis on post-mortem PD brains highlighted a characteristic pattern of small, round RBPs-positive granules in neuronal cell bodies, which also accumulated alongside α- Syn oligomers. Interestingly, different RBPs were found to be involved in Lewy body formation, and quantitative analyses showed a significant RBPs increase in mature compared to undefined aggregates. This suggests that SGs may act as an intermediate for α-Syn aggregation in PD brains. Simultaneously, SH-SY5Y cells exposed to both acute and chronic treatment with the oxidative stress inducer sodium arsenite showed an increase in intracellular aggregates positive for both SGs markers and α-Syn, indicating enhanced recruitment or association of α-Syn with SGs. Future work will aim to explore the impact of both α-Syn aggregation and microtubule dynamics on the homeostasis of SGs in PD pathology, providing further insight into the molecular mechanisms of the disease.

Background/Aimsα-Synucleinopathy in the brain is the neuropathological hallmark of Parkinson’s disease (PD). However, the functional impact of α-synucleinopathy in the enteric nervous system remains unknown. We aim to evaluate the association between gastrointestinal (GI) dysfunction and α-synuclein (αSYN) pathology in the stomach and colon of PD patients and controls, as well as to investigate the association between the αSYN pathology in GI tract and future PD risk.MethodsA total of 35 PD patients and 52 neurologically intact subjects were enrolled in this study. Endoscopic biopsies were performed, and then immunohistochemical staining for αSYN was performed. All subjects completed the validated Rome III questionnaire for the assessment of GI symptoms. The association between GI symptoms and the αSYN pathology in GI mucosa was evaluated. Incident PD cases were assessed during a median follow-up of 46 months.ResultsThe proportion of self-reported constipation and functional constipation through the Rome III questionnaire was significantly higher in PD patients than in controls (P < 0.001 and P = 0.015). However, no significant association was found between the αSYN pathology in the stomach and colon mucosa and constipation, as well as other GI symptoms including dyspepsia symptoms and abdominal discomfort or pain, regardless of the presence or absence of clinical PD (P > 0.05). No incident PD cases were diagnosed during study period.ConclusionsOur present study suggests that the deposition of αSYN in the mucosal enteric nervous system may not be reflected by functional impairment of the affected segment of the gut.