Conference Agenda

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of familial PD (4%) and occur in a significant number (1%) of idiopathic PD cases. Pathogenic mutations of LRRK2, among which the p.G2019S is by far the most frequent, induces an increase of LRRK2 kinase activity, which is currently the target in clinical studies employing LRRK2 kinase inhibitors as disease modifying agents in PD. To investigate whether p.G2019S LRRK2 modulates alpha-synuclein (aSyn) pathology in an age-dependent way, we injected 3- and 12-month-old G2019S KI and wild-type (WT) mice bilaterally in substantia nigra (SN) with adeno-associated viral vectors (AAV2/9) carrying human A53T aSyn, to induce de novo aSyn aggregation via overexpression. G2019S KI mice accumulated larger amount of phosphoSerine129 (pSer129) positive inclusions in striatum and SN compacta (SNc) with respect to WT mice. Strikingly, this difference appeared in older mice and was associated with larger SNc cell loss (Novello et al., Neurobiol Dis 120, 21-33, 2018). To interrogate whether p.G2019S LRRK2 also favors aSyn seeding and spreading in an age-dependent fashion, 3- and 18-month-old G2019S KI and WT mice were injected unilaterally in the striatum with murine aSyn preformed fibrils (PFFs). In young mice, 3 months after PFFs injection, pSer129-positive aSyn inclusions were evident in the striatum, cerebral cortex and, to greater extent in the amygdala regardless of the genotype. Instead, in 18-month-old mice, assessed 1 month after PFF injection, a significantly reduced burden of pSer129-positive aSyn inclusions was observed in the SNc and cerebral cortex of G2019S KI mice compared to WT controls. This correlated with a reduced Iba-1 and CD68 staining, an index of microglial activation, in SNc. We conclude that p.G2019S LRRK2 acts on both early (de novo aggregation) and later (seeding and spreading) phases of aSyn pathology but requires the additional effect of ageing.

Toxic aggregates of α-synuclein (αsyn) are considered key drivers of Parkinson’s disease (PD) pathology. In early PD, αsyn induces synaptic dysfunction also modulating the glutamatergic neurotransmission. Here, we described the role of Rabphilin-3A (Rph3A) as novel target to counteract αsyn-induced synaptic loss in PD. Rph3A is a synaptic protein interacting with αsyn and involved in stabilizing dendritic spines and in promoting the synaptic retention of NMDA-type glutamate receptors. We found that in vivo intrastriatal injection of αsyn-preformed fibrils in mice induces the early loss of striatal synapses associated with decreased synaptic levels of Rph3A and impaired Rph3A/NMDA receptors interaction. Modulating Rph3A striatal expression or interfering with the Rph3A/αsyn complex with a small molecule prevented dendritic spine loss and rescued associated early motor defects in αsyn-injected mice.

Conversely, Rph3A expression at the synapse and its interaction with GluN2A-containing NMDARs were increased in parkinsonian animals displaying levodopa (L-DOPA)-induced dyskinesias (LIDs). Acute treatment of dyskinetic animals with a cell-permeable peptide able to interfere with Rph3A/GluN2A binding significantly reduced their abnormal motor behaviour.

Overall, these findings indicate that approaches aimed at restoring Rph3A synaptic functions can slow down synaptic detrimental effects both in early PD and in LIDs.

The seminal work published in 1964 by Drs. Steele, Richardson, and Olszewski detailing a series of patients with postural instability, ocular motor abnormalities, facial and cervical dystonia, and dementia was the first to describe a “heterogeneous system degeneration” now called Progressive Supranuclear Palsy (PSP). The initial neuropathologic findings described argyrophyllic globose and flame-shaped inclusions in both the gray and white matter throughout the brainstem, subcortical and neocortical regions with neuronal loss and white matter degeneration. Immunohistochemistry studies revealed that these inclusions were accumulations in neurons and glial cells of the microtubule-associated protein TAU in various morphologies. Alternative splicing generates six isoforms of the TAU protein. Treatment is still largely supportive.

In the present study, primary cultures of human fibroblasts were established from 6 patients and 3 controls. PSP fibroblasts present hyperphosphorylated TAU aggregates in the cytosol (peri-nuclear area) and the nucleus and colocalizes with TIA-1 and G3BP1 two stress granule markers. Furthermore, we will provide evidence showing alterations in mitochondrial proteins and the mitochondrial spare respiratory capacity, with striking differences within patients. Herein, we have also investigated TAU interaction with chromatin.

Altogether, our results show molecular defects in the peripheral tissue of patients and suggest that fibroblasts can, therefore, be useful in modeling pathways linked to TAU pathology and PSP neurodegeneration.

Deposition of β-sheet-rich amyloid aggregates composed of disease-specific proteins is a feature shared by many neurodegenerative disorders. Misfolding and aggregation of these proteins play a key role in neurotoxicity. In this context, molecular chaperones are key effectors of proteostasis, assisting protein folding and liaising with degradative pathways. Clusterin (Clu) is a multifunctional protein that owns chaperone-like properties and is highly expressed in the brain. Variations in CLU gene are risk factor for late-onset Alzheimer’s disease. Clu mediates both amyloid beta and tau deposition and clearance, but whether this modulation is neuroprotective or neurotoxic is still controversial. Very recently, Clu has also been associated to the regulation of α-synuclein (αSyn) biology in Parkinson’s disease (PD). On one hand, Clu binds αSyn and inhibits its αSyn aggregation. On the other hand, Clu limited the uptake of αSyn fibrils by astrocytes, thus, possibly contributing to αSyn spreading. In this work we aim to better elucidate the understudied role of Clu in PD pathology in post-mortem human brains using immunohistochemical technique, proximity ligation assay and high-resolution confocal microscopy. First, we confirmed the presence of Clu in Lewy bodies and neurites, and we found extracellular Clu accumulation in PD brains. Moreover, we evaluated whether a differential Clu distribution exists between healthy subjects and PD patients. In particular, we explored Clu expression in different neuronal populations and glial cells. Our preliminary data show a vesicle-shaped staining of Clu in the cytoplasm of nigral neurons, but also astrocytes, and oligodendrocytes surrounding tyrosine hydroxylase-positive fibers. Interestingly, Clu was also present in endothelial cells, and the staining in PD patients resembles amyloid angiopathy. This study will help to clarify the interplay between Clu and PD pathology, that, would be key in the perspective of developing a therapeutic strategy.