Establishing the perineuronal net as a neuroprotective barrier in Parkinson’s disease

Parkinson’s disease (PD) is a progressive neurodegenerative disorder. Disease progression is mirrored by the appearance of Lewy pathology (LP) in the brain, which contains misfolded α-synuclein (αSYN). Transmission of misfolded α-synuclein between neurons is key to LP spread. Determinants of neuronal vulnerability to αSYN transmission have been investigated but factors of neuronal resistance have not. One potential factor is the presence of a perineuronal net (PNN). The PNN is a condensed form of extracellular matrix (ECM) that enwraps specific populations of neurons and regulates plasticity. It is an aggregated structure formed of protein and glycosaminoglycans. The PNN forms and condenses on neuronal membranes, creating a dense and polyanionic structure with a reticular morphology, which could block αSYN transmission. The aim of this thesis is to establish whether the PNN is a neuroprotective barrier conferring resistance to neurons against αSYN seeding and pathology in PD.
To investigate whether the PNN blocks αSYN uptake an in vitro neuronal PNN culture model was established. This model accurately replicated mature cortical PNNs, both in terms of the heterogeneity in PNN composition and its maturation. PNNs transitioned from an immature punctate morphology to the reticular morphology as observed in the mature CNS. We also observed a small population of PNNs that were mature at an earlier time point and a distinct composition, highlighting further heterogeneity. We have established a primary culture model of PNN of use to the PNN field.
To investigate the barrier function of PNNs to αSYN, we first purified αSYN and created two, distinct aggregated αSYN species: oligomers and preformed fibrils (PFFs). DIV56 neuronal cultures, containing mature PNNs, were treated with either with 7 µM Alexa 488 labelled oligomer and uptake measured 24 hours later by immunofluorescence or with PFFs and cultured for a further 21 days. The presence of a PNN reduced the uptake of αSYN oligomer in neurons by two thirds (Oligomer positive: PNN positive 15.8 ± 2.52% versus PNN negative: 49.9 ± 2.95%, one-way ANOVA, p<0.05). PNN removal by chondroitinase ABC (ChABC) abolished resistance, significantly increasing oligomer positive neurons (PNN intact: 53.4 ± 2.54% vs degraded: 67.1%, two sample t Test, p<0.05). 70 nM PFF treatment caused progressive accumulation of phosphorylated-αSYN staining over 21 days of treatment, reminiscent of αSYN pathology. Enzymatic removal of the PNN before PFF treatment significantly increased p-αSYN intensity (D21: PNN intact: 0.00231 ± 0.00047 RFU vs degraded 0.00141 ± 0.00047, one-way ANOVA, p<0.05). Using surface plasmon resonance, we have highlighted a potential mechanism for the neuroprotection: αSYN species were found to bind to PNN glycosaminoglycans- chondroitin sulphate E and heparin sulphate. We investigated whether PNN populations were spared in PD brains to establish the relevance of the neuroprotection mechanism. In all brain regions, PNN densities in PD brains were not significantly different compared to non-demented controls (t test, p>0.05). Furthermore, no LP bearing PNN neurons were observed. Together this indicates that PNN populations are unaffected in PD brains, demonstrating that PNN-mediated neuroprotection is relevant in PD. This thesis has established that the PNN is a neuroprotective barrier in PD and protects neurons from developing αSYN pathology in vitro and in PD brains. This opens a new research avenue in pursuit of disease halting therapies in PD.