Talene A. Yacoubian

Parkinson’s Disease (PD) and other synucleinopathies are characterized by the formation of inclusions comprised of alpha-synuclein (αsyn) among

14-3-3 proteins impact protein-protein interactions (PPIs) that regulate neuronal functions. The 14-3-3θ isoform is protective in models of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Human PD and DLB brains show increased 14-3-3θ phosphorylation at S232. To understand the impact of 14-3-3θ phosphorylation on brain PPIs, we performed affinity purification-mass spectrometry using S232 phospho-mutant knock-in mouse models. Proteins binding 14-3-3θ in Cre control cortical lysates were enriched in proteins involved in neuronal morphogenesis and microtubule dynamics. We found a dramatic decrease in proteins binding to 14-3-3θ in S232D mice compared to S232A mice. Axonal trafficking associated with these differentially binding proteins. Live imaging of acidic vesicles in axons revealed reduced net velocity in S232A and S232D neurons compared to that in Cre controls. In S232D neurons, this was due to a dramatic increase in vesicle pausing, while S232A neurons showed reduced segmental velocity, suggesting disrupted dynein motility. We conclude that 14-3-3θ phosphorylation fine tunes axonal transport of acidic vesicles. Disruption of axonal transport with aberrant phosphorylation observed in PD and DLB could contribute to impaired clearance of aggregated proteins in these disorders.

Microglia, the resident immune cells of the central nervous system (CNS), are involved in the pathogenesis of neurodegenerative diseases, such as Alzheimer's disease (AD), Dementia with Lewy Bodies (DLB), and Parkinson's disease (PD). 14‐3‐3 proteins act as molecular hubs to regulate protein–protein interactions, which are involved in numerous cellular functions, including cellular signaling, protein folding, and apoptosis. We previously revealed decreased 14‐3‐3 levels in the brains of human subjects with neurodegenerative diseases. In this study, we examined the role of 14‐3‐3 proteins in the microglial proinflammatory response to lipopolysaccharide (LPS). We found that LPS treatment induced 14‐3‐3 protein levels within 6 hours. With the use of BV02 and dimeric fourteen‐three‐three peptide inhibitor (difopein), a small molecule and peptide inhibitor of 14‐3‐3 protein–protein interactions, respectively, we found a dramatic increase in microglial activation markers in both immortalized BV‐2 microglial cells and in primary mouse microglia. Both 14‐3‐3 inhibitors also increased LPS‐induced microglial phagocytosis, lysosomal proteolysis, and cytokine release in primary microglia. In contrast, chemotaxis toward the cellular damage stimulus, adenosine triphosphate (ATP), was diminished with 14‐3‐3 inhibition. Inhibition of 14‐3‐3's hastened LPS‐induced activation of the nuclear factor‐kB (NF‐κB) signaling pathway, as measured by its nuclear translocation. 14‐3‐3's reduced activation of the NF‐κB pathway by binding and inhibiting the release of IκB kinase beta (IKKβ). Disruption of 14‐3‐3's binding to IKKβ with BV02 or difopein increased the downstream phosphorylation and degradation of the inhibitor of NF‐κB alpha (IκBα). Collectively, our findings suggest 14‐3‐3 proteins play a critical role in the regulation of inflammatory responses in microglia and may serve as potential targets for immunotherapy of CNS diseases. image

Objective: To examine if immune cell changes are present in early stages of Dementia with Lewy Bodies (DLB).Background: Evidence for inflammation in synucleinopathies has been building. An extensive number of studies have documented inflammatory changes in Parkinson’s disease (PD), but much less is known about DLB. Post-mortem DLB brains show T-cell infiltration, and flow cytometry and cytokine/chemokine measures in DLB subjects have suggested the presence of inflammatory responses in DLB. However, there is limited understanding if immune cell changes are present at the earliest stages of DLB.Methods: We recruited a small, exploratory cohort of nine subjects with Mild Cognitive Impairment—Lewy Bodies (MCI-LB) as part of the broader Alabama Udall cohort study, which is focused on the role of inflammation in early PD. The MCI-LB cohort was compared to subjects with de novo PD (n = 64) and healthy controls (n = 70) from the larger Alabama Udall cohort. Subjects underwent clinical assessments, including Movement Disorder Society-United Parkinson’s Disease rating scale (MDS-UPDRS), and comprehensive cognitive assessment. Blood was obtained for flow cytometry and cytokine/chemokine analyses.Results: Demographics and medical history were comparable between groups. Significant differences in University of Pennsylvania Smell Identification Test, Schwab and England Activities of Daily Living, and MDS-UPDRS scores were observed between the groups. MCI-LB subjects had poorer cognitive composite scores compared to PD participants. MCI-LB subjects demonstrated a robust 60% reduction in mature neutrophils compared to healthy controls.Conclusion: Our data show changes in innate immune cells in MCI-LB compared to healthy controls in this small exploratory study. A larger cohort study is needed to validate these findings.

14-3-3 proteins, particularly the 14-3-3θ isoform, are neuroprotective in several models of Parkinson's disease (PD). Evidence for increased 14-3-3θ phosphorylation observed in PD and other neurodegenerative diseases points to a possible pathogenic role for 14-3-3θ phosphorylation in neurodegenerative disease. We recently created a novel conditional knock-in mouse to express the 14-3-3θ S232D phosphomimetic mutation. After crossing this conditional knock-in mouse with the Emx1-Cre mouse in order to induce expression of the S232D mutation in the cortex and hippocampus, we evaluated the effect of 14-3-3θ phosphorylation on behavior and pathology. These mice demonstrated mild motor deficits and reduced social dominance behavior but showed normal cognition and anxiety levels compared to Cre control mice. S232D mice did not show any α-synuclein or phospho-tau pathology at baseline, and dendritic arborization was normal in primary hippocampal cultures from S232D mice. Overall, this mouse model is a novel tool that can be used to look at the effect of 14-3-3θ phosphorylation at S232 in the context of neurodegenerative disease models.

Alpha-synuclein (αsyn) is the key pathogenic protein implicated in synucleinopathies including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). In these diseases, αsyn is thought to spread between cells where it accumulates and induces pathology; however, mechanisms that drive its propagation or aggregation are poorly understood. We have previously reported that the small GTPase Rab27b is elevated in human PD and DLB and that it can mediate the autophagic clearance and toxicity of αsyn in a paracrine αsyn cell culture model. Here, we expanded our previous work and characterized the role of Rab27b in neuronal lysosomal processing and αsyn clearance. We found that Rab27b KD in this αsyn-inducible neuronal model resulted in lysosomal dysfunction and increased αsyn levels in lysosomes. Similar lysosomal proteolytic defects and enzymatic dysfunction were observed in both primary neuronal cultures and brain lysates from male and female Rab27b knock-out (KO) mice. αSyn aggregation was exacerbated in Rab27b KO neurons upon treatment with αsyn preformed fibrils. We found no changes in lysosomal counts or lysosomal pH in either model, but we did identify changes in acidic vesicle trafficking and in lysosomal enzyme maturation and localization, which may drive lysosomal dysfunction and promote αsyn aggregation. Rab27b OE enhanced lysosomal activity and reduced insoluble αsyn accumulation. Finally we found elevated Rab27b levels in human postmortem incidental Lewy body disease subjects relative to healthy controls. These data suggest the role of Rab27b in neuronal lysosomal activity and identify it as a potential therapeutic target in synucleinopathies.

Alzheimer’s (AD) and Parkinson disease (PD) pathology often co-occur. Amyloid-β and phosphorylated tau are found in 30-50% of idiopathic PD cases, while α-synuclein inclusions are present in 50% of AD cases. These co-pathologies are linked to increased mortality and earlier onset of cognitive decline. Immune activation is a hallmark of these neurodegenerative diseases, but current models primarily examine each pathology in isolation. How these co-pathologies drive inflammation and neuronal loss remains poorly understood. We therefore developed a mouse model combining tau, amyloid-β, and α-synuclein. We found that co-pathologies synergistically trigger an amplified neuroimmune response, with expanded populations of CD4 ⁺ and CD8 ⁺ tissue-resident memory T cells and CD68 ⁺ microglia, compared to single pathologies. These changes were abundant in the hippocampus and cortex, regions with elevated protein pathology load and enhanced neuronal loss. Our findings demonstrate that co-pathologies enhance proteinopathy and synergistically enhance immune activation and neurodegeneration, suggesting that combinatorial therapeutic strategies that target both co-pathologies and inflammation, may be disease modifying. Summary: Webster et al. demonstrate that co-occurring Alzheimer’s and Parkinson disease protein pathologies, common in cognitively impaired patient populations, amplify proteinopathy and synergistically enhance CNS neuroinflammatory responses and neurodegeneration. This work supports the need for combinatorial therapeutic strategies and positions neuroinflammation as an important link for co-pathology enhanced neurodegeneration.

LRRK2 mutations are among the most common genetic causes for Parkinson’s disease (PD), and toxicity is associated with increased kinase activity. 14-3-3 proteins are key interactors that regulate LRRK2 kinase activity. Phosphorylation of the 14-3-3θ isoform at S232 is dramatically increased in human PD brains. Here we investigate the impact of 14-3-3θ phosphorylation on its ability to regulate LRRK2 kinase activity. Both wildtype and the non-phosphorylatable S232A 14-3-3θ mutant reduced the kinase activity of wildtype and G2019S LRRK2, whereas the phosphomimetic S232D 14-3-3θ mutant had minimal effects on LRRK2 kinase activity, as determined by measuring autophosphorylation at S1292 and T1503 and Rab10 phosphorylation. However, wildtype and both 14-3-3θ mutants similarly reduced the kinase activity of the R1441G LRRK2 mutant. 14-3-3θ phosphorylation did not promote global dissociation with LRRK2, as determined by co-immunoprecipitation and proximal ligation assays. 14-3-3s interact with LRRK2 at several phosphorylated serine/threonine sites, including T2524 in the C-terminal helix, which can fold back to regulate the kinase domain. Interaction between 14-3-3θ and phosphorylated T2524 LRRK2 was important for 14-3-3θ’s ability to regulate kinase activity, as wildtype and S232A 14-3-3θ failed to reduce the kinase activity of G2019S/T2524A LRRK2. Finally, we found that the S232D mutation failed to protect against G2019S LRRK2-induced neurite shortening in primary cultures, while the S232A mutation was protective. We conclude that 14-3-3θ phosphorylation destabilizes the interaction of 14-3-3θ with LRRK2 at T2524, which consequently promotes LRRK2 kinase activity and toxicity.