Closing the structure-to-function gap for LRRK2.

Variations in the LRRK2 gene represent one of the strongest genetic factors for Parkinson's disease (PD). It has become clear that structural knowledge of the encoded large multidomain LRRK2 protein will cast light on its biological function. The new study from Myasnikov, Zhu, et al. provides a high-resolution structure of the full-length LRRK2.

Genetic association of primary nonresponse to anti-TNFα therapy in patients with inflammatory bowel disease.

OBJECTIVES: Primary nonresponse (PNR) to antitumor necrosis factor-α (TNFα) biologics is a serious concern in patients with inflammatory bowel disease (IBD). We aimed to identify the genetic variants associated with PNR. PATIENTS AND METHODS: Patients were recruited from outpatient GI clinics and PNR was determined using both clinical and endoscopic findings. A case-control genome-wide association study was performed in 589 IBD patients and associations were replicated in an independent cohort of 293 patients. Effect of the associated variant on gene expression and TNFα secretion was assessed by cell-based assays. Pleiotropic effects were investigated by Phenome-wide association study (PheWAS). RESULTS: We identified rs34767465 as associated with PNR to anti-TNFα therapy (odds ratio: 2.07, 95% CI, 1.46-2.94, P = 2.43 × 10-7, [replication odds ratio: 1.8, 95% CI, 1.04-3.16, P = 0.03]). rs34767465 is a multiple-tissue expression quantitative trait loci for FAM114A2. Using RNA-sequencing and protein quantification from HapMap lymphoblastoid cell lines (LCLs), we found a significant decrease in FAM114A2 mRNA and protein expression in both heterozygous and homozygous genotypes when compared to wild type LCLs. TNFα secretion was significantly higher in THP-1 cells [differentiated into macrophages] with FAM114A2 knockdown versus controls. Immunoblotting experiments showed that depletion of FAM114A2 impaired autophagy-related pathway genes suggesting autophagy-mediated TNFα secretion as a potential mechanism. PheWAS showed rs34767465 was associated with comorbid conditions found in IBD patients (derangement of joints [P = 3.7 × 10-4], pigmentary iris degeneration [P = 5.9 × 10-4], diverticulum of esophagus [P = 7 × 10-4]). CONCLUSIONS: We identified a variant rs34767465 associated with PNR to anti-TNFα biologics, which increases TNFα secretion through mechanism related to autophagy. rs34767465 may also explain the comorbidities associated with IBD.

Glucocerebrosidase haploinsufficiency in A53T α-synuclein mice impacts disease onset and course.

Mutations in GBA1 encountered in Gaucher disease are a leading risk factor for Parkinson disease and associated Lewy body disorders. Many GBA1 mutation carriers, especially those with severe or null GBA1 alleles, have earlier and more progressive parkinsonism. To model the effect of partial glucocerebrosidase deficiency on neurological progression in vivo, mice with a human A53T α-synuclein (SNCAA53T) transgene were crossed with heterozygous null gba mice (gba+/-). Survival analysis of 84 mice showed that in gba+/-//SNCAA53T hemizygotes and homozygotes, the symptom onset was significantly earlier than in gba+/+//SNCAA53T mice (p-values 0.023-0.0030), with exacerbated disease progression (p-value <0.0001). Over-expression of SNCAA53T had no effect on glucocerebrosidase levels or activity. Immunoblotting demonstrated that gba haploinsufficiency did not lead to increased levels of either monomeric SNCA or insoluble high molecular weight SNCA in this model. Immunohistochemical analyses demonstrated that the abundance and distribution of SNCA pathology was also unaltered by gba haploinsufficiency. Thus, while the underlying mechanism is not clear, this model shows that gba deficiency impacts the age of onset and disease duration in aged SNCAA53T mice, providing a valuable resource to identify modifiers, pathways and possible moonlighting roles of glucocerebrosidase in Parkinson pathogenesis.

(G2019S) LRRK2 causes early-phase dysfunction of SNpc dopaminergic neurons and impairment of corticostriatal long-term depression in the PD transgenic mouse.

Twelve- to sixteen-month-old (G2019S) LRRK2 transgenic mice prepared by us displayed progressive neuronal death of substantia nigra pars compacta (SNpc) dopaminergic cells. In the present study, we hypothesized that prior to a late-phase death of SNpc dopaminergic neurons, (G2019S) LRRK2 also causes an early-phase neuronal dysfunction of SNpc dopaminergic cells in the (G2019S) LRRK2 mouse. Eight to nine-month-old (G2019S) LRRK2 transgenic mice exhibited the symptom of hypoactivity in the absence of the degeneration of SNpc dopaminergic neurons or nigrostriatal dopaminergic terminals. Whole-cell current-clamp recordings of SNpc dopaminergic cells in brain slices demonstrated a significant decrease in spontaneous firing frequency of SNpc dopaminergic neurons of 8-month-old (G2019S) LRRK2 mice. Carbon fiber electrode amperometry recording using striatal slices showed that (G2019S) LRRK2 transgenic mice at the age of 8 to 9months display an impaired evoked dopamine release in the dorsolateral striatum. Normal nigrostriatal dopaminergic transmission is required for the induction of long-term synaptic plasticity expressed at corticostriatal glutamatergic synapses of striatal medium spiny neurons. Whole-cell voltage-clamp recordings showed that in contrast to medium spiny neurons of 8 to 9-month-old wild-type mice, high-frequency stimulation of corticostriatal afferents failed to induce long-term depression (LTD) of corticostriatal EPSCs in medium spiny neurons of (G2019S) LRRK2 mice at the same age. Our study provides the evidence that mutant (G2019S) LRRK2 causes early-phase dysfunctions of SNpc dopaminergic neurons, including a decrease in spontaneous firing rate and a reduction in evoked dopamine release, and impairment of corticostriatal LTD in the (G2019S) LRRK2 transgenic mouse.

LRRK2 mediates haloperidol-induced changes in indirect pathway striatal projection neurons.

Haloperidol is used to manage psychotic symptoms in several neurological disorders through mechanisms that involve antagonism of dopamine D2 receptors that are highly expressed in the striatum. Significant side effects of haloperidol, known as extrapyramidal symptoms, lead to motor deficits similar to those seen in Parkinson's disease and present a major challenge in clinical settings. The underlying molecular mechanisms responsible for these side effects remain poorly understood. Parkinson's disease-associated LRRK2 kinase has an important role in striatal physiology and a known link to dopamine D2 receptor signaling. Here, we systematically explore convergent signaling of haloperidol and LRRK2 through pharmacological or genetic inhibition of LRRK2 kinase, as well as knock-in mouse models expressing pathogenic mutant LRRK2 with increased kinase activity. Behavioral assays show that LRRK2 kinase inhibition ameliorates haloperidol-induced motor changes in mice. A combination of electrophysiological and anatomical approaches reveals that LRRK2 kinase inhibition interferes with haloperidol-induced changes, specifically in striatal neurons of the indirect pathway. Proteomic studies and targeted intracellular pathway analyses demonstrate that haloperidol induces a similar pattern of intracellular signaling as increased LRRK2 kinase activity. Our study suggests that LRRK2 kinase plays a key role in striatal dopamine D2 receptor signaling underlying the undesirable motor side effects of haloperidol. This work opens up new therapeutic avenues for dopamine-related disorders, such as psychosis, also furthering our understanding of Parkinson's disease pathophysiology.

Molecular and spatial transcriptomic classification of midbrain dopamine neurons and their alterations in a LRRK2G2019S model of Parkinson's disease.

Several studies have revealed that midbrain dopamine (DA) neurons, even within a single neuroanatomical area, display heterogeneous properties. In parallel, studies using single cell profiling techniques have begun to cluster DA neurons into subtypes based on their molecular signatures. Recent work has shown that molecularly defined DA subtypes within the substantia nigra (SNc) display distinctive anatomic and functional properties, and differential vulnerability in Parkinson's disease (PD). Based on these provocative results, a granular understanding of these putative subtypes and their alterations in PD models, is imperative. We developed an optimized pipeline for single-nuclear RNA sequencing (snRNA-seq) and generated a high-resolution hierarchically organized map revealing 20 molecularly distinct DA neuron subtypes belonging to three main families. We integrated this data with spatial MERFISH technology to map, with high definition, the location of these subtypes in the mouse midbrain, revealing heterogeneity even within neuroanatomical sub-structures. Finally, we demonstrate that in the preclinical LRRK2G2019S knock-in mouse model of PD, subtype organization and proportions are preserved. Transcriptional alterations occur in many subtypes including those localized to the ventral tier SNc, where differential expression is observed in synaptic pathways, which might account for previously described DA release deficits in this model. Our work provides an advancement of current taxonomic schemes of the mouse midbrain DA neuron subtypes, a high-resolution view of their spatial locations, and their alterations in a prodromal mouse model of PD.

A novel antagonist of the CCL5/CCR5 axis suppresses the tumor growth and metastasis of triple-negative breast cancer by CCR5-YAP1 regulation.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer (BC) with a high mortality rate, and few effective therapeutic strategies are available. CCL5/CCR5 is an appealing immunotherapeutic target for TNBC. However, its signaling mechanism is poorly understood and its direct antagonists have not been reported. Here, we developed a high-throughput screening (HTS) assay for discovering its antagonists. Verteporfin was identified as a more selective and potent antagonist than the known CCR5 antagonist maraviroc. Without photodynamic therapy, verteporfin demonstrated significant inhibition on TNBC tumor growth through immune regulation, remarkable suppression of lung metastasis by cell-intrinsic mechanism, and a significant extension of overall survival in vivo. Mechanistically, CCR5 was found to be essential for expression of the key hippo effector YAP1. It promoted YAP1 transcription via HIF-1α and exerted further control over the migration of CD8+ T, NK, and MDSC immune cells through chemokines CXCL16 and CXCL8 which were identified from RNA-seq. Moreover, the CCR5-YAP1 axis played a vital role in promoting metastasis by modulating ß-catenin and core epithelial-mesenchymal transition transcription factors ZEB1 and ZEB2. It is noteworthy that the regulatory relationship between CCR5 and YAP1 was observed across various BC subtypes, TNBC patients, and showed potential relevance in fifteen additional cancer types. Overall, this study introduced an easy-to-use HTS assay that streamlines the discovery of CCL5/CCR5 axis antagonists. Verteporfin was identified as a specific molecular probe of this axis with great potentials as a therapeutic agent for treating sixteen malignant diseases characterized by heightened CCR5 and YAP1 levels.

R1441C and G2019S LRRK2 knockin mice have distinct striatal molecular, physiological, and behavioral alterations.

LRRK2 mutations are closely associated with Parkinson's disease (PD). Convergent evidence suggests that LRRK2 regulates striatal function. Here, by using knock-in mouse lines expressing the two most common LRRK2 pathogenic mutations-G2019S and R1441C-we investigated how LRRK2 mutations altered striatal physiology. While we found that both R1441C and G2019S mice displayed reduced nigrostriatal dopamine release, hypoexcitability in indirect-pathway striatal projection neurons, and alterations associated with an impaired striatal-dependent motor learning were observed only in the R1441C mice. We also showed that increased synaptic PKA activities in the R1441C and not G2019S mice underlie the specific alterations in motor learning deficits in the R1441C mice. In summary, our data argue that LRRK2 mutations' impact on the striatum cannot be simply generalized. Instead, alterations in electrochemical, electrophysiological, molecular, and behavioral levels were distinct between LRRK2 mutations. Our findings offer mechanistic insights for devising and optimizing treatment strategies for PD patients.

A radiomics-based model for predicting local control of resected brain metastases receiving adjuvant SRS.

PURPOSE: Adjuvant radiosurgery to the cavities of surgically resected brain metastases provides excellent local tumor control while reducing the risk of deleterious cognitive decline associated with whole brain radiotherapy. A subset of these patients, however, will develop disease recurrence following radiosurgery. In this study, we sought to assess the predictive capability of radiomic-based models, as compared with standard clinical features, in predicting local tumor control. METHODS: We performed a retrospective chart review of patients treated with adjuvant radiosurgery for resected brain metastases at the "Institution" from 2009 to 2019. Shape, intensity and texture based radiomics features of the cavities were extracted from the pre-radiosurgery treatment planning MRI scans and trained using a gradient boosting technique with K-fold cross validation. RESULTS: In total, 71 cavities from 67 treated patients were included for analysis. The 6 and 12 month local control estimates were 86% and 76%, respectively. The 6 and 12 month overall survival was 78% and 55%, respectively. Thirty-six patients developed intracranial failures outside of the surgical cavity. The predictive model for local control trained on imaging features from the whole cavity achieved an area-under-the-curve (AUC) of 0.73 on the validation set versus an AUC of 0.40 for the clinical features. CONCLUSIONS: Here we report a single institutional experience using radiomic-based predictive modeling of local tumor control following adjuvant Gamma Knife radiosurgery for resected brain metastases. We found the radiomics features to provide more robust predictive models of local control rates versus clinical features alone. Such techniques could potentially prove useful in the clinical setting and warrant further investigation.

Pathway-specific dysregulation of striatal excitatory synapses by LRRK2 mutations.

LRRK2 is a kinase expressed in striatal spiny projection neurons (SPNs), cells which lose dopaminergic input in Parkinson's disease (PD). R1441C and G2019S are the most common pathogenic mutations of LRRK2. How these mutations alter the structure and function of individual synapses on direct and indirect pathway SPNs is unknown and may reveal pre-clinical changes in dopamine-recipient neurons that predispose toward disease. Here, R1441C and G2019S knock-in mice enabled thorough evaluation of dendritic spines and synapses on pathway-identified SPNs. Biochemical synaptic preparations and super-resolution imaging revealed increased levels and altered organization of glutamatergic AMPA receptors in LRRK2 mutants. Relatedly, decreased frequency of miniature excitatory post-synaptic currents accompanied changes in dendritic spine nano-architecture, and single-synapse currents, evaluated using two-photon glutamate uncaging. Overall, LRRK2 mutations reshaped synaptic structure and function, an effect exaggerated in R1441C dSPNs. These data open the possibility of new neuroprotective therapies aimed at SPN synapse function, prior to disease onset.

Parkinson's disease is caused by progressive damage to regions of the brain that regulate movement. This leads to a loss in nerve cells that produce a signaling molecule called dopamine, and causes patients to experience shakiness, slow movement and stiffness. When dopamine is released, it travels to a part of the brain known as the striatum, where it is received by cells called spiny projection neurons (SPNs), which are rich in a protein called LRRK2. Mutations in this protein have been shown to cause the motor impairments associated with Parkinson's disease. SPNs send signals to other regions of the brain either via a 'direct' route, which promotes movement, or an 'indirect' route, which suppresses movement. Previous studies suggest that mutations in the gene for LRRK2 influence the activity of these pathways even before dopamine signaling has been lost. Yet, it remained unclear how different mutations independently affected each pathway. To investigate this further, Chen et al. studied two of the mutations most commonly found in the human gene for LRRK2, known as G2019S and R1441C. This involved introducing one of these mutations in to the genetic code of mice, and using fluorescent proteins to mark single SPNs in either the direct or indirect pathway. The experiments showed that both mutations disrupted the connections between SPNs in the direct and indirect pathway, which altered the activity of nerve cells in the striatum. Chen et al. found that individual connections were more strongly affected by the R1441C mutation. Further experiments showed that this was caused by the re-organization of a receptor protein in the nerve cells of the direct pathway, which increased how SPNs responded to inputs from other nerve cells. These findings suggest that LRRK2 mutations disrupt neural activity in the striatum before dopamine levels become depleted. This discovery could help researchers identify new therapies for treating the early stages of Parkinson's disease before the symptoms of dopamine loss arise.

NQO1 regulates mitotic progression and response to mitotic stress through modulating SIRT2 activity.

Previous studies have shown that SIRT2 plays a role in mitosis through deacetylating specific downstream targets. However, the upstream regulation of SIRT2 activity has been relatively unexplored. In this study, we provide evidence that NAD(P)H:quinone oxidoreductase 1 (NQO1) interacts with and activates SIRT2 in an NAD-dependent manner. Strong protein-protein interaction and co-localization of the two proteins during mitosis is required to maintain an active NQO1-SIRT2 axis which is critical for successful completion of mitosis. This is evident by the observed delay in mitotic exit in cells upon NQO1 inhibition. Mechanistically, this phenotype can be explained by the decrease in APC/C complex activity resulting from decreased SIRT2 deacetylation activity. Furthermore, we show that this newly established role of NQO1 has an impact on how cancer cells may respond to mitotic stress. In this regard, both pharmacologic and genetic NQO1 inhibition increases sensitivity to anti-mitotic drugs functioning as microtubule poisons by inducing mitotic arrest and allowing cells to accumulate cell death signals. Therefore, the significant prognostic value of NQO1 in predicting outcome of cancer patients might be explained in part due to the functional contribution of NQO1-SIRT2 axis to mitotic stress. Altogether, this novel mechanism of action further supports the pleiotropic biological effects exerted by NQO1 in addition to its antioxidant function and it might provide the basis for expanding the therapeutic potential of NQO1 inhibition towards increasing sensitivity to standard treatments.

(R1441C) LRRK2 induces the degeneration of SN dopaminergic neurons and alters the expression of genes regulating neuronal survival in a transgenic mouse model.

Mutation of leucine-rich repeat kinase 2 (LRRK2) is the most common genetic cause of both familial and sporadic Parkinson's disease (PD) cases. Several mutations in LRRK2 gene were reported in PD patients. R1441 is the second most frequent site of LRRK2 mutation. We generated (R1441C) LRRK2 transgenic mice that displayed motor deficits at the age of 16 months. Compared with wild-type mice, 16-month-old (R1441C) LRRK2 mice exhibited a significant reduction in the number of substantia nigra (SN) dopaminergic neurons. To elucidate molecular pathogenic pathways involved in (R1441C) LRRK2-induced death of SN dopaminergic neurons, we performed microarray analysis to visualize altered mRNA expressions in the SN of (R1441C) LRRK2 mouse. In the SN of (R1441C) LRRK2 transgenic mouse, the mRNA expression of three genes that promote cell death was upregulated, while the mRNA expression of seven genes that contribute to neurogenesis/neuroprotection was significantly downregulated. Our results suggest that altered expression of these genes involved in regulating neuronal survival may contribute to the pathogenesis of (R1441C) LRRK2-induced PD.