Abstract Body

Dementia with Lewy bodies (DLB) is characterized by mixed pathology, Lewy bodies and neurites as well as concomitant AD pathology. This heterogeneity is reflected in a variable and frequently non-specific clinical picture. Mutations in the GBA gene are frequent among patients with DLB worldwide however among Ashkenazi Jewish patients with DLB, the prevalence of GBA mutations is very high and is approximately 33%. Mutations in the GBA gene appear to have multiple and varied effects on clinical presentation as well as on pathology - both of which are an important focus of research worldwide. In order to improve the accuracy of diagnosis and subsequent clinical management, the genetic factors that influence disease need to be better understood. In this lecture I will present recent work from our institution and others, which have examined the impact of GBA mutations and several other common genetic factors on the development and progression of DLB.

Aims

LRRK2 mutations are the most common genetic cause of Parkinson’s disease but the pathogenic mechanism is unclear. We aim to investigate the normal physiological role of LRRK2 and the pathogenic mechanisms underlying LRRK2 mutations.

Methods

We use genetic, biochemical, histological, ultrastructural, behavioral, and neurochemical approaches to determine the consequences of LRRK inactivation or LRRK2 R1441C and G2019S mutations using knockout (KO) and knockin (KI) mice.

Results

We found that inactivation of LRRK1/2 results in impaired motor coordination and loss of dopaminergic (DA) terminals in the striatum, followed by DA neuron death in the substantia nigra. Evoked dopamine release is also reduced in the striatum of LRRK-null mice. By 20 and 25 months of age, LRRK-null mice develop more severe DA neurodegeneration, but the cerebral cortex is still unaffected. However, the R1441C KI alleles are unable to support DA neuron survival, as indicated by a significant reduction of DA neurons in LRRK1-/-; LRRK2 R1441C KI/KI mice at 20 and 25 months of age, though DA neurodegeneration is not as severe as that in LRRK-null mice. Surprisingly, in contrast to R1441C, the G2019S KI alleles are able to support DA neuron survival, as indicated by normal number of DA neurons in LRRK1-/-; LRRK2 G2019S KI/KI mice at 20 and 25 months of age.

Conclusions

Our genetic findings demonstrate that the LRRK family plays an essential role in support of DA neuron survival during aging and show that LRRK2 R1441C and G2019S mutations differ in their consequences on DA neuron survival in the aging mouse brain.

Aims

Elevated leucine rich repeat kinase 2 (LRRK2) activity contributes to familial and idiopathic Parkinson’s disease (PD), as does reduction of beta-glucocerebrosidase (Gcase) function. Emerging data suggests LRRK2 kinase activity disrupts lysosomal function. We investigated if increased LRRK2 activity reduces Gcase function, a lysosomal enzyme, or alters accumulation of Gcase substrates, glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph).

Methods

Gcase activity was assessed using the artificial substrate 4-MUG, and a novel, highly sensitive LC-MS/MS-based assay quantified GlcSph and GlcCer lipids in two murine models carrying PD-associated mutations: Lrrk2 G2019S and Gba D409V knock-in lines

Results

A cross-sectional study revealed brain GlcSph levels were significantly increased in 12 and 16-month (mo) Lrrk2 G2019S homozygous (HOM) mice compared to wild-type (WT). Brain GlcCer was also significantly elevated in 16mo G2019S HOM mice but Gcase activity was not altered between genotypes at any age. Chronic treatment with MLi-2, a potent, selective, and brain-penetrant LRRK2 kinase inhibitor, normalized brain GlcSph in 14mo HOM mice to WT levels. Chronic MLi-2 treatment also significantly reduced brain GlcSph in D409V HOM, but not HET mice. Plasma GlcSph and GlcCer were significantly reduced in MLi-2 treated D409V HOM and HET.

Conclusions

These data support that LRRK2 kinase activity influences accumulation of Gcase lipid substrates in the brain, particularly GlcSph. Gcase activity and lipid levels are currently being analyzed in brain tissues, CSF, and plasma from PD subjects with LRRK2 or GBA mutations alongside idiopathic PD and controls. These studies aim to advance biomarker and therapeutic strategies for PD patients.

Aims

Autophagic-lysosomal dysfunction is at the centre of Parkinson’s disease (PD), although the precise pathological mechanisms remain unknown. Recent evidence has shown that expression of LRRK2-G2019S or hWT-LRRK2 inhibited autophagosome production, whereas LRRK2-R1441C shows impaired autolysosome fusion and increased lysosomal pH. In parallel, mitochondrial defects have been reported in LRRK2 related PD, such as altered mitochondria morphology and homeostasis. At the interface of these two pathways, mitophagy is also impaired in LRRK2-G2019S. The aim of our study is to further investigate the impact of LRRK2 mutations (G2019S and R1441C) on the autophagy-lysosomal pathway, mitophagy and mitochondrial function.

Methods

The aim of our study is to further investigate the impact of LRRK2 mutations (G2019S and R1441C) on the autophagy-lysosomal pathway, mitophagy and mitochondrial function, and identify a mechanism for the observed phenotypes using cortical primary cultures and iPSC-derived dopaminergic neurons. Furthermore, we investigate the potential of MLi-2, a LRRK2 kinase inhibitor, and Clioquinol, a zinc ionophore that can restore lysosomal function in LRRK2 related defects. To characterise these pathways we aim to use a combination of functional assays with immunoblotting and immunocytochemistry.

Results

In our study we show altered lysosomal-autophagy function and impaired mitochondrial function in LRRK2-G2019S and LRRK2-R1441C in both primary cortical neurons and iPSC-DA neurons. We also show the effect of MLi-2 and Clioquinol on these pathways.

Conclusions

This could elucidate how autophagy-lysosomal and mitochondrial deficits are related to PD. Ultimately the outcome of this study could have an impact on PD treatment, focusing on novel drug approaches to correct lysosomal and mitochondrial dysfunction.

Aims

As gene–environment interactions strongly impact Parkinson’s disease (PD) risk, we set out to develop an in vivo model of nigrostriatal dopaminergic (DA) neurodegeneration involving LRRK2 vulnerability, ageing and inflammation, three factors critical for PD

Methods

To address the role of the PD-linked LRRK2-G2019S mutation in regulating age-dependant immune responses and nigrostriatal DA vulnerability, we exposed WT-LRRK2 and G2019S-LRRK2 transgenic mice to low-grade chronic inflammation induced by intraperitoneal injection of lipopolysaccharide (LPS) twice weekly over the mouse life span. We selected two age groups- 3-4 M (young adult) and 7-8M (middle-aged)- to start LPS injections

Results

LPS treatment progressively reduced motor coordination in G2019S-LRRK2 mice compared to WT-LRRK2. In the substantia nigra pars compacta (SNpc) of G2019S-LRRK2 mice treated with LPS, we detected a 60-70% decrease of tyrosine hydroxylase-positive neurons at 10-12 months, when compared to WT-LRRK2 mice of the same age and treatment. Furthermore, a dramatic astro/microgliosis as well as increased RC2+/IBA1- and infiltrating peripheral CD3+ cells were observed in the G2019S-LRRK2 SNpc. In parallel, a significant up-regulation of inflammatory markers, including pro-IL-1β, IL-1β, and Casp1, was detected in LRRK2*G2019S compared to WT-LPS mice, suggesting LRRK2-mediated inflammasome over-activation. At the peripheral level, G2019S-LRRK2 splenic macrophages from LPS-treated mice revealed a significant higher production of proinflammatory cytokines

Conclusions

Current findings indicate that the synergy between aging, LRRK2-G2019S, and low-grade inflammation promotes neurodegeneration. This novel model will enable the identification of key interactors playing roles in DA neurodegeneration, both at the central and peripheral level

Aims

Methods

Results

Conclusions