Molecular characterisation of autophagy deficits in a LRRK2-BAC transgenic rat model of Parkinsonâs disease

Parkinsonâs disease (PD) is the second most common neurodegenerative disorder worldwide. PD is characterised by the preferential loss of dopaminergic neurons in the Substantia Nigra pars compacta in the midbrain accompanied by progressive motor dysfunction. The precise aetiology of PD is unknown, however a causative role of Leucine-rich repeat kinase 2 (LRRK2) has been proposed. Mutations in the LRRK2 gene are the most frequent cause of familial PD and are also an independent risk factor for sporadic PD. Although the function of LRRK2 is not well characterised, a role of LRRK2 in the autophagy pathway has been suggested. The disruption of the autophagy pathway by LRRK2 pathogenic mutations has been described. However, the literature is often contradictory and the exact underlying mechanisms remain unknown. In this study, primary cortical cultures were generated from three bacterial artificial chromosome (BAC) transgenic (TG) rat models of PD harbouring either the whole human wild-type LRRK2 gene, the G2019S mutant (the most common LRRK2 mutation) or the R1441C mutant (the LRRK2 mutation leading to a more aggressive pathology). After characterising the autophagy pathway it was observed that the presence of either hWT-LRRK2 or LRRK2-G2019S inhibits autophagosome biogenesis in primary cortical cultures. hWT-LRRK2 and LRRK2-G2019S each localise to the Golgi apparatus where autophagy signalling complexes are situated, which may underlie the inhibitory effect on autophagosome biogenesis. The presence of LRRK2-R1441C, however, induces a lysosomal deficit that causes an accumulation in autophagosomes and decreased autolysosome maturation and lysosomal protein degradation. LRRK2-R1441C also increases lysosomal pH levels and causes lysosomal Ca2+ release deficits. Furthermore, hWT-LRRK2 and LRRK2 G2019S were found to bind to the a1 subunit of the v-type ATPase pump (vATPase a1) which is responsible for modulating lysosomal pH. Whereas LRRK2-R1441C showed a loss of binding capacity to this subunit, which was associated with a decrease in a1 subunit protein expression and cellular mislocalisation. Lastly, the Zn2+ ionophore, clioquinol, was able to rescue the LRRK2-R1441C-mediated lysosomal phenotypes through modulating lysosomal zinc levels and increased vATPase a1 expression which re-acidified lysosomes, corrected localised calcium release and increased autolysosome maturation. These data describe a novel functional link between LRRK2 and vATPase a1, and define critical mechanisms underlying the inhibition of autophagy by different pathogenic LRRK2 mutations. These findings demonstrate a novel mode of action by which drugs may rescue lysosomal dysfunction and alleviate blockage in autophagic flux. These results demonstrate the importance of LRRK2 in lysosomal biology, as well as the critical role of the lysosome in PD and the potential of lysosome-targeting compounds as a novel therapeutic for the disease.</p

Molecular characterisation of autophagy deficits in a LRRK2-BAC transgenic rat model of Parkinson’s disease

Parkinson’s disease (PD) is the second most common neurodegenerative disorder worldwide. PD is characterised by the preferential loss of dopaminergic neurons in the Substantia Nigra pars compacta in the midbrain accompanied by progressive motor dysfunction. The precise aetiology of PD is unknown, however a causative role of Leucine-rich repeat kinase 2 (LRRK2) has been proposed. Mutations in the LRRK2 gene are the most frequent cause of familial PD and are also an independent risk factor for sporadic PD. Although the function of LRRK2 is not well characterised, a role of LRRK2 in the autophagy pathway has been suggested. The disruption of the autophagy pathway by LRRK2 pathogenic mutations has been described. However, the literature is often contradictory and the exact underlying mechanisms remain unknown. In this study, primary cortical cultures were generated from three bacterial artificial chromosome (BAC) transgenic (TG) rat models of PD harbouring either the whole human wild-type LRRK2 gene, the G2019S mutant (the most common LRRK2 mutation) or the R1441C mutant (the LRRK2 mutation leading to a more aggressive pathology). After characterising the autophagy pathway it was observed that the presence of either hWT-LRRK2 or LRRK2-G2019S inhibits autophagosome biogenesis in primary cortical cultures. hWT-LRRK2 and LRRK2-G2019S each localise to the Golgi apparatus where autophagy signalling complexes are situated, which may underlie the inhibitory effect on autophagosome biogenesis. The presence of LRRK2-R1441C, however, induces a lysosomal deficit that causes an accumulation in autophagosomes and decreased autolysosome maturation and lysosomal protein degradation. LRRK2-R1441C also increases lysosomal pH levels and causes lysosomal Ca2+ release deficits. Furthermore, hWT-LRRK2 and LRRK2 G2019S were found to bind to the a1 subunit of the v-type ATPase pump (vATPase a1) which is responsible for modulating lysosomal pH. Whereas LRRK2-R1441C showed a loss of binding capacity to this subunit, which was associated with a decrease in a1 subunit protein expression and cellular mislocalisation. Lastly, the Zn2+ ionophore, clioquinol, was able to rescue the LRRK2-R1441C-mediated lysosomal phenotypes through modulating lysosomal zinc levels and increased vATPase a1 expression which re-acidified lysosomes, corrected localised calcium release and increased autolysosome maturation. These data describe a novel functional link between LRRK2 and vATPase a1, and define critical mechanisms underlying the inhibition of autophagy by different pathogenic LRRK2 mutations. These findings demonstrate a novel mode of action by which drugs may rescue lysosomal dysfunction and alleviate blockage in autophagic flux. These results demonstrate the importance of LRRK2 in lysosomal biology, as well as the critical role of the lysosome in PD and the potential of lysosome-targeting compounds as a novel therapeutic for the disease.</p

LRRK2 BAC transgenic rats develop progressive, L-DOPA-responsive motor impairment, and deficits in dopamine circuit function

Mutations in leucine-rich repeat kinase 2 (LRRK2) lead to late-onset, autosomal dominant Parkinson's disease, characterized by the degeneration of dopamine neurons of the substantia nigra pars compacta, a deficit in dopamine neurotransmission and the development of motor and non-motor symptoms. The most prevalent Parkinson's disease LRRK2 mutations are located in the kinase (G2019S) and GTPase (R1441C) encoding domains of LRRK2. To better understand the sequence of events that lead to progressive neurophysiological deficits in vulnerable neurons and circuits in Parkinson's disease, we have generated LRRK2 bacterial artificial chromosome transgenic rats expressing either G2019S or R1441C mutant, or wild-type LRRK2, from the complete human LRRK2 genomic locus, including endogenous promoter and regulatory regions. Aged (18–21 months) G2019S and R1441C mutant transgenic rats exhibit L-DOPA-responsive motor dysfunction, impaired striatal dopamine release as determined by fast-scan cyclic voltammetry, and cognitive deficits. In addition, in vivo recordings of identified substantia nigra pars compacta dopamine neurons in R1441C LRRK2 transgenic rats reveal an age-dependent reduction in burst firing, which likely results in further reductions to striatal dopamine release. These alterations to dopamine circuit function occur in the absence of neurodegeneration or abnormal protein accumulation within the substantia nigra pars compacta, suggesting that nigrostriatal dopamine dysfunction precedes detectable protein aggregation and cell death in the development of Parkinson's disease. In conclusion, our longitudinal deep-phenotyping provides novel insights into how the genetic burden arising from human mutant LRRK2 manifests as early pathophysiological changes to dopamine circuit function and highlights a potential model for testing Parkinson's therapeutics

Gene therapy restores dopamine transporter expression and ameliorates pathology in iPSC and mouse models of infantile parkinsonism

Most inherited neurodegenerative disorders are incurable, and often only palliative treatment is available. Precision medicine has great potential to address this unmet clinical need. We explored this paradigm in dopamine transporter deficiency syndrome (DTDS), caused by biallelic loss-of-function mutations in SLC6A3, encoding the dopamine transporter (DAT). Patients present with early infantile hyperkinesia, severe progressive childhood parkinsonism, and raised cerebrospinal fluid dopamine metabolites. The absence of effective treatments and relentless disease course frequently leads to death in childhood. Using patient-derived induced pluripotent stem cells (iPSCs), we generated a midbrain dopaminergic (mDA) neuron model of DTDS that exhibited marked impairment of DAT activity, apoptotic neurodegeneration associated with TNFα-mediated inflammation, and dopamine toxicity. Partial restoration of DAT activity by the pharmacochaperone pifithrin-μ was mutation-specific. In contrast, lentiviral gene transfer of wild-type human SLC6A3 complementary DNA restored DAT activity and prevented neurodegeneration in all patient-derived mDA lines. To progress toward clinical translation, we used the knockout mouse model of DTDS that recapitulates human disease, exhibiting parkinsonism features, including tremor, bradykinesia, and premature death. Neonatal intracerebroventricular injection of human SLC6A3 using an adeno-associated virus (AAV) vector provided neuronal expression of human DAT, which ameliorated motor phenotype, life span, and neuronal survival in the substantia nigra and striatum, although off-target neurotoxic effects were seen at higher dosage. These were avoided with stereotactic delivery of AAV2.SLC6A3 gene therapy targeted to the midbrain of adult knockout mice, which rescued both motor phenotype and neurodegeneration, suggesting that targeted AAV gene therapy might be effective for patients with DTDS

APOE and immunity: Research highlights

INTRODUCTION: At the Alzheimer's Association's APOE and Immunity virtual conference, held in October 2021, leading neuroscience experts shared recent research advances on and inspiring insights into the various roles that both the apolipoprotein E gene (APOE) and facets of immunity play in neurodegenerative diseases, including Alzheimer's disease and other dementias. METHODS: The meeting brought together more than 1200 registered attendees from 62 different countries, representing the realms of academia and industry. RESULTS: During the 4-day meeting, presenters illuminated aspects of the cross-talk between APOE and immunity, with a focus on the roles of microglia, triggering receptor expressed on myeloid cells 2 (TREM2), and components of inflammation (e.g., tumor necrosis factor α [TNFα]). DISCUSSION: This manuscript emphasizes the importance of diversity in current and future research and presents an integrated view of innate immune functions in Alzheimer's disease as well as related promising directions in drug development