Dysregulated mRNA Translation in the G2019S LRRK2 and LRRK2 Knock-Out Mouse Brains.

The G2019S mutation in leucine-rich repeat kinase 2 (LRRK2) causes familial Parkinson's disease (PD) and is also found in a subset of idiopathic cases. Prior studies in Drosophila and human induced pluripotent stem cell (iPSC)-derived dopamine neurons uncovered a pronounced effect of G2019S LRRK2 on mRNA translation. It was previously reported that G2019S LRRK2 promotes translation of mRNAs with complex 5' untranslated region (UTR) secondary structure, resulting in increased expression of calcium channels and dysregulated calcium homeostasis in human dopamine neurons. Here, we show that dysregulated translation occurs in the brains of mammalian LRRK2 models in vivo Through ribosome profiling studies of global translation, we observe that mRNAs with complex 5'UTR structure are also preferentially translated in the G2019S LRRK2-expressing mouse brain. Reporter assays suggest that this 5'UTR preference is independent of translation initiation factors. Conversely, translation of mRNAs with complex 5'UTR secondary structure is downregulated in LRRK2 knock-out (KO) mouse brain, indicating a robust link between LRRK2 kinase activity and translation of mRNA with complex 5'UTR structure. Further, substantia nigra pars compacta (SNpc) dopamine neurons in the G2019S LRRK2-expressing brain exhibit increased calcium influx, which is consistent with the previous report from human dopamine neurons. These results collectively suggest that LRRK2 plays a mechanistic role in translational regulation, and the G2019S mutation in LRRK2 causes translational defects leading to calcium dysregulation in the mammalian brain.

Protocol for measurement of calcium dysregulation in human induced pluripotent stem cell-derived dopaminergic neurons.

Calcium regulation is a critical process in neurons, and Ca2+ signaling is a major contributor to neurological disorders including Parkinson's disease (PD). Here, combining calcium imaging with whole-cell Ca2+ current recording, we provide a detailed protocol for measuring Ca2+ homeostasis in dopaminergic (DA) neurons derived from human induced pluripotent stem cells (hiPSCs). This approach can be applied to investigate the role of Ca2+ homeostasis in neuronal functionality as well as in disease processes. For complete details on the use and execution of this protocol, please refer to Kim et al. (2020).

Defects in mRNA Translation in LRRK2-Mutant hiPSC-Derived Dopaminergic Neurons Lead to Dysregulated Calcium Homeostasis.

The G2019S mutation in leucine-rich repeat kinase 2 (LRRK2) is a common cause of familial Parkinson's disease (PD). This mutation results in dopaminergic neurodegeneration via dysregulated protein translation, although how alterations in protein synthesis contribute to neurodegeneration in human neurons is not known. Here we define the translational landscape in LRRK2-mutant dopaminergic neurons derived from human induced pluripotent stem cells (hiPSCs) via ribosome profiling. We found that mRNAs that have complex secondary structure in the 5' untranslated region (UTR) are translated more efficiently in G2019S LRRK2 neurons. This leads to the enhanced translation of multiple genes involved in Ca2+ regulation and to increased Ca2+ influx and elevated intracellular Ca2+ levels, a major contributor to PD pathogenesis. This study reveals a link between dysregulated translation control and Ca2+ homeostasis in G2019S LRRK2 human dopamine neurons, which potentially contributes to the progressive and selective dopaminergic neurotoxicity in PD.

Meta-Analysis of the Alzheimer's Disease Human Brain Transcriptome and Functional Dissection in Mouse Models.

We present a consensus atlas of the human brain transcriptome in Alzheimer's disease (AD), based on meta-analysis of differential gene expression in 2,114 postmortem samples. We discover 30 brain coexpression modules from seven regions as the major source of AD transcriptional perturbations. We next examine overlap with 251 brain differentially expressed gene sets from mouse models of AD and other neurodegenerative disorders. Human-mouse overlaps highlight responses to amyloid versus tau pathology and reveal age- and sex-dependent expression signatures for disease progression. Human coexpression modules enriched for neuronal and/or microglial genes broadly overlap with mouse models of AD, Huntington's disease, amyotrophic lateral sclerosis, and aging. Other human coexpression modules, including those implicated in proteostasis, are not activated in AD models but rather following other, unexpected genetic manipulations. Our results comprise a cross-species resource, highlighting transcriptional networks altered by human brain pathophysiology and identifying correspondences with mouse models for AD preclinical studies.

PINK1 Primes Parkin-Mediated Ubiquitination of PARIS in Dopaminergic Neuronal Survival.

Mutations in PTEN-induced putative kinase 1 (PINK1) and parkin cause autosomal-recessive Parkinson's disease through a common pathway involving mitochondrial quality control. Parkin inactivation leads to accumulation of the parkin interacting substrate (PARIS, ZNF746) that plays an important role in dopamine cell loss through repression of proliferator-activated receptor gamma coactivator-1-alpha (PGC-1α) promoter activity. Here, we show that PARIS links PINK1 and parkin in a common pathway that regulates dopaminergic neuron survival. PINK1 interacts with and phosphorylates serines 322 and 613 of PARIS to control its ubiquitination and clearance by parkin. PINK1 phosphorylation of PARIS alleviates PARIS toxicity, as well as repression of PGC-1α promoter activity. Conditional knockdown of PINK1 in adult mouse brains leads to a progressive loss of dopaminergic neurons in the substantia nigra that is dependent on PARIS. Altogether, these results uncover a function of PINK1 to direct parkin-PARIS-regulated PGC-1α expression and dopaminergic neuronal survival.

LRRK2 pathobiology in Parkinson's disease - virtual inclusion.

A common cause of Parkinson disease are missense mutations in the leucine-rich repeat kinase 2 (LRRK2) catalytic Roc-COR domain, leading to a decrease in GTPase activity; and its kinase domain, leading to an increase in kinase activity and subsequent LRRK2 toxicity. Targeting LRRK2 with selective, brain-permeable kinase inhibitors is a promising approach to reduce toxicity, and thus is a major goal of clinical development. Understanding the specific signaling cascades triggered by LRRK2 mutations will be key to this aim. This article is part of a special issue on Parkinson disease.

Abberant protein synthesis in G2019S LRRK2 Drosophila Parkinson disease-related phenotypes.

LRRK2 mutations are a frequent cause of familial Parkinson disease (PD) and are also found in a number of sporadic PD cases. PD-linked G2019S and I2020T mutations in the kinase domain of LRRK2 result in elevated kinase activity, which is required for the toxicity of these pathogenic variants in cell and animal models of PD. We recently reported that LRRK2 interacts with and phosphorylates a number of mammalian ribosomal proteins, several of which exhibit increased phosphorylation via both G2019S and I2020T LRRK2. Blocking the phosphorylation of ribosomal protein s15 through expression of phospho-deficient T136A s15 prevents age-associated locomotor deficits and dopamine neuron loss caused by G2019S LRRK2 expression in Drosophila indicating that s15 is a pathogenic LRRK2 substrate. We previously described that G2019S LRRK2 causes an induction of bulk mRNA translation that is blocked by T136A s15 or the protein synthesis inhibitor anisomycin. Here, we report the protective effects of the eIF4E/eIF4G interaction inhibitor 4EGI-1, in preventing neurodegenerative phenotypes in G2019S LRRK2 flies, and discuss how our findings and those of other groups provide a framework to begin investigating the mechanistic impact of LRRK2 on translation.

LRRK2 pathobiology in Parkinson's disease.

Mutations in the catalytic Roc-COR and kinase domains of leucine-rich repeat kinase 2 (LRRK2) are a common cause of familial Parkinson's disease (PD). LRRK2 mutations cause PD with age-related penetrance and clinical features identical to late-onset sporadic PD. Biochemical studies support an increase in LRRK2 kinase activity and a decrease in GTPase activity for kinase domain and Roc-COR mutations, respectively. Strong evidence exists that LRRK2 toxicity is kinase dependent leading to extensive efforts to identify selective and brain-permeable LRRK2 kinase inhibitors for clinical development. Cell and animal models of PD indicate that LRRK2 mutations affect vesicular trafficking, autophagy, protein synthesis, and cytoskeletal function. Although some of these biological functions are affected consistently by most disease-linked mutations, others are not and it remains currently unclear how mutations that produce variable effects on LRRK2 biochemistry and function all commonly result in the degeneration and death of dopamine neurons. LRRK2 is typically present in Lewy bodies and its toxicity in mammalian models appears to be dependent on the presence of α-synuclein, which is elevated in human iPS-derived dopamine neurons from patients harboring LRRK2 mutations. Here, we summarize biochemical and functional studies of LRRK2 and its mutations and focus on aberrant vesicular trafficking and protein synthesis as two leading mechanisms underlying LRRK2-linked disease.

Ribosomal protein s15 phosphorylation mediates LRRK2 neurodegeneration in Parkinson's disease.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of familial and sporadic Parkinson's disease (PD). Elevated LRRK2 kinase activity and neurodegeneration are linked, but the phosphosubstrate that connects LRRK2 kinase activity to neurodegeneration is not known. Here, we show that ribosomal protein s15 is a key pathogenic LRRK2 substrate in Drosophila and human neuron PD models. Phosphodeficient s15 carrying a threonine 136 to alanine substitution rescues dopamine neuron degeneration and age-related locomotor deficits in G2019S LRRK2 transgenic Drosophila and substantially reduces G2019S LRRK2-mediated neurite loss and cell death in human dopamine and cortical neurons. Remarkably, pathogenic LRRK2 stimulates both cap-dependent and cap-independent mRNA translation and induces a bulk increase in protein synthesis in Drosophila, which can be prevented by phosphodeficient T136A s15. These results reveal a novel mechanism of PD pathogenesis linked to elevated LRRK2 kinase activity and aberrant protein synthesis in vivo.