Identification of Targets from LRRK2 Rescue Phenotypes.

Parkinson's disease (PD) is an age-dependent neurodegenerative condition. Leucine-rich repeat kinase 2 (LRRK2) mutations are the most frequent cause of sporadic and autosomal dominant PD. The exact role of LRRK2 protective variants (R1398H, N551K) together with a pathogenic mutant (G2019S) in aging and neurodegeneration is unknown. We generated the following myc-tagged UAS-LRRK2 transgenic Drosophila: LRRK2 (WT), N551K, R1398H, G2019S single allele, and double-mutants (N551K/G2019S or R1398H/G2019S). The protective variants alone were able to suppress the phenotypic effects caused by the pathogenic LRRK2 mutation. Next, we conducted RNA-sequencing using mRNA isolated from dopaminergic neurons of these different groups of transgenic Drosophila. Using pathway enrichment analysis, we identified the top 10 modules (p < 0.05), with "LRRK2 in neurons in Parkinson's disease" among the candidates. Further dissection of this pathway identified the most significantly modulated gene nodes such as eEF1A2, ACTB, eEF1A, and actin cytoskeleton reorganization. The induction of the pathway was successfully restored by the R1398H protective variant and R1398H-G2019S or N551K-G2019S rescue experiments. The oxidoreductase family of genes was also active in the pathogenic mutant and restored in protective and rescue variants. In summary, we provide in vivo evidence supporting the neuroprotective effects of LRRK2 variants. RNA sequencing of dopaminergic neurons identified upregulation of specific gene pathways in the Drosophila carrying the pathogenic variant, and this was restored in the rescue phenotypes. Using protective gene variants, our study identifies potential new targets and provides proof of principle of a new therapeutic approach that will further our understanding of aging and neurodegeneration in PD.

Chetomin rescues pathogenic phenotype of LRRK2 mutation in drosophila.

Leucine-rich repeat kinase 2 (LRRK2) is a complex protein kinase involved in a diverse set of functions. Mutations in LRRK2 are a common cause of autosomal dominant familial Parkinson's disease. Peroxiredoxin 2 (PRDX2) belongs to a family of anti-oxidants that protect cells from oxidative stress. Importantly, PRDX2 is a cytoplasmic protein, similar to Leucine-rich repeat kinase 2, which localizes predominantly in the cytosol. Here, we demonstrated that Leurice-rich repeat kinase 2 phosphorylates PRDX2 in Drosophila, leading to a loss of dopaminergic neurons, climbing ability and shortened lifespan. These pathogenic phenotypes in the LRRK2 Drosophila were rescued with transgenic expression of PRDX2. Chetomin, a PRDX2 mimic, belongs to a class of epidithio-diketopiperazine fungal secondary metabolites (containing a dithiol group that has hydrogen peroxide-reducing activity). As proof of principle, we demonstrated that Chetomin recapitulated the rescue in these mutant Drosophila. Our findings suggest that Chetomin can be a potential therapeutic compound in LRRK2 linked Parkinson's disease.

Retinal Vessel Analysis as a Novel Screening Tool to Identify Childhood Acute Lymphoblastic Leukemia Survivors at Risk of Cardiovascular Disease.

BACKGROUND: Microvascular endothelial dysfunction is central to the pathogenesis of cardiovascular disease (CVD). The eye offers direct access for endothelial health assessment via the retinal microvasculature. The aim of the study was to investigate whether image-based retinal vessel analysis is a feasible method of assessing endothelial health in survivors of childhood acute lymphoblastic leukemia (cALL). MATERIALS AND METHODS: Cardiovascular risk factors (CRFs) were estimated using the 30-year Framingham Risk Score in 73 childhood leukemia survivors (median age: 25; median years from diagnosis: 19) and 78 healthy controls (median age: 23). Radial arterial stiffness was measured using pulse wave analyzer, while endothelial activation markers were measured by soluble intercellular adhesion molecule 1 (sICAM-1) and soluble vascular cell adhesion molecule 1 (sVCAM-1). Retinal fundus images were analyzed for central retinal artery/vein equivalents (CRAE/CRVE) and arteriolar-venular ratio (AVR). RESULTS: cALL survivors had higher CRF (P<0.0001), arterial stiffness (P=0.001), and sVCAM-1 (P=0.007) compared with controls. Survivors also had significantly higher CRVE (P=0.021) while AVR was significantly lower (P=0.026) in survivors compared with controls, compatible with endothelial dysfunction. In cALL survivors with intermediate risk for CVD, CRAE, and AVR are significantly lower, while sVCAM-1 and sICAM-1 are significantly higher when compared with survivors with low CVD risk after adjusting with covariates (age, sex, and smoking status). CONCLUSIONS: cALL survivors have an increased risk of CVD compared with age-matched peers. The survivors demonstrated microvasculopathy, as measured by retinal vascular analysis, in addition to physical and biochemical evidence of endothelial dysfunction. These changes predate other measures of CVD. Retinal vessel analysis may be utilized as a robust screening tool for identifying survivors at increased risk for developing CVD.

Temporal changes in gut microbiota profile in children with acute lymphoblastic leukemia prior to commencement-, during-, and post-cessation of chemotherapy.

BACKGROUND: Alteration in gut microbiota has been recently linked with childhood leukemia and the use of chemotherapy. Whether the perturbed microbiota community is restored after disease remission and cessation of cancer treatment has not been evaluated. This study examines the chronological changes of gut microbiota in children with acute lymphoblastic leukemia (ALL) prior to the start-, during-, and following cessation of chemotherapy. METHODOLOGY: We conducted a longitudinal observational study in gut microbiota profile in a group of paediatric patients diagnosed with ALL using 16 s ribosomal RNA sequencing and compared these patients' microbiota pattern with age and ethnicity-matched healthy children. Temporal changes of gut microbiota in these patients with ALL were also examined at different time-points in relation to chemotherapy. RESULTS: Prior to commencement of chemotherapy, gut microbiota in children with ALL had larger inter-individual variability compared to healthy controls and was enriched with bacteria belonging to Bacteroidetes phylum and Bacteroides genus. The relative abundance of Bacteroides decreased upon commencement of chemotherapy. Restitution of gut microbiota composition to resemble that of healthy controls occurred after cessation of chemotherapy. However, the microbiota composition (beta diversity) remained distinctive and a few bacteria were different in abundance among the patients with ALL compared to controls despite completion of chemotherapy and presumed restoration of normal health. CONCLUSION: Our findings in this pilot study is the first to suggest that gut microbiota profile in children with ALL remains marginally different from healthy controls even after cessation of chemotherapy. These persistent microbiota changes may have a role in the long-term wellbeing in childhood cancer survivors but the impact of these changes in subsequent health perturbations in these survivors remain unexplored.

Linking the effects of helminth infection, diet and the gut microbiota with human whole-blood signatures.

Helminth infection and dietary intake can affect the intestinal microbiota, as well as the immune system. Here we analyzed the relationship between fecal microbiota and blood profiles of indigenous Malaysians, referred to locally as Orang Asli, in comparison to urban participants from the capital city of Malaysia, Kuala Lumpur. We found that helminth infections had a larger effect on gut microbial composition than did dietary intake or blood profiles. Trichuris trichiura infection intensity also had the strongest association with blood transcriptional profiles. By characterizing paired longitudinal samples collected before and after deworming treatment, we determined that changes in serum zinc and iron levels among the Orang Asli were driven by changes in helminth infection status, independent of dietary metal intake. Serum zinc and iron levels were associated with changes in the abundance of several microbial taxa. Hence, there is considerable interplay between helminths, micronutrients and the microbiota on the regulation of immune responses in humans.

Enrichment of gut-derived Fusobacterium is associated with suboptimal immune recovery in HIV-infected individuals.

We explored the gut microbiota profile among HIV-infected individuals with diverse immune recovery profiles following long-term suppressive ART and investigated the relationship between the altered bacteria with markers of immune dysfunction. The microbiota profile of rectal swabs from 26 HIV-infected individuals and 20 HIV-uninfected controls were examined. Patients were classified as suboptimal responders, sIR (n = 10, CD4 T-cell <350 cells/ul) and optimal responders, oIR (n = 16, CD4 T-cell >500 cells/ul) after a minimum of 2 years on suppressive ART. Canonical correlation analysis(CCA) and multiple regression modelling were used to explore the association between fecal bacterial taxa abundance and immunological profiles in optimal and suboptimal responders. We found Fusobacterium was significantly enriched among the HIV-infected and the sIR group. CCA results showed that Fusobacterium abundance was negatively correlated with CD4 T-cell counts, but positively correlated with CD4 T-cell activation and CD4 Tregs. Multiple linear regression analysis adjusted for age, baseline CD4 T-cell count, antibiotic exposure and MSM status indicated that higher Fusobacterium relative abundance was independently associated with poorer CD4 T-cell recovery following ART. Enrichment of Fusobacterium was associated with reduced immune recovery and persistent immune dysfunction following ART. Modulating the abundance of this bacterial taxa in the gut may be a viable intervention to improve immune reconstitution in our setting.

Phosphorylation of amyloid precursor protein by mutant LRRK2 promotes AICD activity and neurotoxicity in Parkinson's disease.

Mutations in LRRK2, which encodes leucine-rich repeat kinase 2, are the most common genetic cause of familial and sporadic Parkinson's disease (PD), a degenerative disease of the central nervous system that causes impaired motor function and, in advanced stages, dementia. Dementia is a common symptom of another neurodegenerative disease, Alzheimer's disease, and research suggests that there may be pathophysiological and genetic links between the two diseases. Aggregates of ß amyloid [a protein produced through cleavage of amyloid precursor protein (APP)] are seen in both diseases and in PD patients carrying G2019S-mutant LRRK2. Using patient-derived cells, brain tissue, and PD model mice, we found that LRRK2 interacted with and phosphorylated APP at Thr668 within its intracellular domain (AICD). Phosphorylation of APP at Thr668 promoted AICD transcriptional activity and correlated with increased nuclear abundance of AICD and decreased abundance of a dopaminergic neuron marker in cultures and brain tissue. The AICD regulates the transcription of genes involved in cytoskeletal dynamics and apoptosis. Overexpression of AICD, but not a phosphodeficient mutant (AICDT668A), increased the loss of dopaminergic neurons in older mice expressing LRRK2G2019S Moreover, the amount of Thr668-phosphorylated APP was substantially greater in postmortem brain tissue and dopaminergic neurons (generated by reprogramming skin cells) from LRRK2G2019S patients than in those from healthy individuals. LRRK2 inhibitors reduced the phosphorylation of APP at Thr668 in the patient-derived dopaminergic neurons and in the midbrains of LRRK2G2019S mice. Thus, APP is a substrate of LRRK2, and its phosphorylation promotes AICD function and neurotoxicity in PD.

Reduced microbial diversity in adult survivors of childhood acute lymphoblastic leukemia and microbial associations with increased immune activation.

BACKGROUND: Adult survivors of childhood cancers such as acute lymphoblastic leukemia (ALL) have health problems that persist or develop years after cessation of therapy. These late effects include chronic inflammation-related comorbidities such as obesity and type 2 diabetes, but the underlying cause is poorly understood. RESULTS: We compared the anal microbiota composition of adult survivors of childhood ALL (N = 73) with healthy control subjects (N = 61). We identified an altered community with reduced microbial diversity in cancer survivors, who also exhibit signs of immune dysregulation including increased T cell activation and chronic inflammation. The bacterial community among cancer survivors was enriched for Actinobacteria (e.g. genus Corynebacterium) and depleted of Faecalibacterium, correlating with plasma concentrations of IL-6 and CRP and HLA-DR+CD4+ and HLA-DR+CD8+ T cells, which are established markers of inflammation and immune activation. CONCLUSIONS: We demonstrated a relationship between microbial dysbiosis and immune dysregulation in adult ALL survivors. These observations suggest that interventions that could restore microbial diversity may ameliorate chronic inflammation and, consequently, development of late effects of childhood cancer survivors.

Immunity in young adult survivors of childhood leukemia is similar to the elderly rather than age-matched controls: Role of cytomegalovirus.

Many treatment complications that occur late in childhood cancer survivors resemble age-related comorbidities observed in the elderly. An immune phenotype characterized by increased immune activation, systemic inflammation, and accumulation of late-differentiated memory CD57(+) CD28(-) T cells has been associated with comorbidities in the elderly. Here, we explored if this phenotype was present in young adult leukemia survivors following an average of 19 years from chemotherapy and/or radiotherapy completion, and compared this with that in age-matched controls. We found that markers of systemic inflammation-IL-6 and human C-reactive protein and immune activation-CD38 and HLA-DR on T cells, soluble CD (sCD)163 from monocytes and macrophages-were increased in survivors compared to controls. T-cell responses specific to cytomegalovirus (CMV) were also increased in survivors compared to controls while CMV IgG levels in survivors were comparable to levels measured in the elderly (>50years) and correlated with IL-6, human C-reactive protein, sCD163, and CD57(+) CD28(-) memory T cells. Immune activation and inflammation markers correlated poorly with prior chemotherapy and radiotherapy exposure. These data suggest that CMV infection/reactivation is strongly correlated with the immunological phenotype seen in young childhood leukemia survivors and these changes may be associated with the early onset of age-related comorbidities in this group.

MAP1B rescues LRRK2 mutant-mediated cytotoxicity.

Leucine-rich repeat kinase 2 (LRRK2) mutations are the most common cause of dominant and sporadic Parkinson's disease (PD), a common neurodegenerative disorder. Yeast-two-hybrid screening using human LRRK2 kinase domain as bait identified microtubule associated protein 1B (MAP1B) as a LRRK2 interactor. The interacting domains were LRRK2 kinase and the light chain portion of MAP1B (LC1). LRRK2 + LC1 interaction resulted in LRRK2 kinase inhibition. LRRK2 mutants (R1441C, G2019S and I2020T) exhibited decreased endogenous LC1 expression and its co-expression with LC1 rescued LRRK2 mutant-mediated toxicity. This study presented the first data on the effects of LRRK2 + LC1 interaction and also suggested that LCI possibly rescued LRRK2 mutant-induced cytotoxicity by inhibiting LRRK2 kinase activity. Compounds that upregulate LC1 expression may therefore hold therapeutic potential for LRRK2-linked diseases.

Thiol peroxidases ameliorate LRRK2 mutant-induced mitochondrial and dopaminergic neuronal degeneration in Drosophila.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are common causes of familial Parkinson's disease (PD). LRRK2 has been shown to bind peroxiredoxin-3 (PRDX3), the most important scavenger of hydrogen peroxide in the mitochondria, in vitro. Here, we examined the interactions of LRRK2 and PRDX3 in Drosophila models by crossing transgenic LRRK2 and PRDX3 flies. As proof of principle experiments, we subsequently challenged LRRK2 and LRRK2/PRDX3 flies with a peroxidase mimic, Ebselen. We demonstrated that co-expression of PRDX3 with the LRRK2 kinase mutant G2019S in bigenic Drosophila ameliorated the G2019S mutant-induced reduction in peroxidase capacity, loss of dopaminergic neurons, shortened lifespan and mitochondrial defects of flight muscles in monogenic flies expressing the G2019S alone. Challenges with Ebselen recapitulated similar rescue of these phenotypic features in mutant-expressing Drosophila. The peroxidase mimic preserved neuronal and mitochondrial and neuronal integrity and improved mobility and survival in mutant-expressing Drosophila. Taken together, our study provides the first in vivo evidence to suggest that phosphoinhibition of endogenous peroxidases could be a mechanism in LRRK2-induced oxidant-mediated neurotoxicity. Our therapeutic experiments also highlight the potential of thiol peroxidases as neuroprotective agents in PD patients carrying LRRK2 mutations.

Similarity of molecular phenotype between known epilepsy gene LGI1 and disease candidate gene LGI2.

BACKGROUND: The LGI2 (leucine-rich, glioma inactivated 2) gene, a prime candidate for partial epilepsy with pericentral spikes, belongs to a family encoding secreted, beta-propeller domain proteins with EPTP/EAR epilepsy-associated repeats. In another family member, LGI1 (leucine-rich, glioma inactivated 1) mutations are responsible for autosomal dominant lateral temporal epilepsy (ADLTE). Because a few LGI1 disease mutations described in the literature cause secretion failure, we experimentally analyzed the secretion efficiency and subcellular localization of several LGI1 and LGI2 mutant proteins corresponding to observed non-synonymous single nucleotide polymorphisms (nsSNPs) affecting the signal peptide, the leucine-rich repeats and the EAR propeller. RESULTS: Mapping of disease-causing mutations in the EAR domain region onto a 3D-structure model shows that many of these mutations co-localize at an evolutionary conserved surface region of the propeller. We find that wild-type LGI2 is secreted to the extracellular medium in glycosylated form similarly to LGI1, whereas several mutant proteins tested in this study are secretion-deficient and accumulate in the endoplasmic reticulum. Interestingly, mutations at structurally homologous positions in the EAR domain have the same effect on secretion in LGI1 and LGI2. CONCLUSIONS: This similarity of experimental mislocalization phenotypes for mutations at homologous positions of LGI2 and the established epilepsy gene LGI1 suggests that both genes share a potentially common molecular pathogenesis mechanism that might be the reason for genotypically distinct but phenotypically related forms of epilepsy.

Is LGI2 the candidate gene for partial epilepsy with pericentral spikes?

Partial epilepsy with pericentral spikes (PEPS) is a familial epilepsy with disease locus mapped to human chromosome region 4p15; yet, the causative gene is unknown. In this work, arguments based on protein sequence analysis and patient-specific chromosomal deletions are provided for LGI2 as the prime candidate gene for PEPS among the 52 genes known at the genome locus 4p15. Furthermore, we suggest that two reports of patients that were not classified as PEPS but show very similar phenotypes and deletions in the PEPS disease locus, could in fact describe the same disease. To test this hypothesis, patients with diagnosed PEPS or the described similar phenotypes could be screened for mutations in LGI2 and other shortlisted candidate genes. The linkage between PEPS and its disease causing gene(s) would allow diagnosis of the disease based on genetic screening as well as hereditary studies. Furthermore, previous knowledge on molecular disease mechanisms of related LGI proteins, for example LGI1 and autosomal dominant lateral temporal epilepsy, could be applied to deepen the understanding of the PEPS disease mechanism at the molecular level, which may facilitate therapeutic intervention in the future. Supplementary Table is available at http://www.worldscinet.com/jbcb/.

Negative regulation of the actin-regulating kinase Prk1p by patch localization-induced autophosphorylation.

The Prk1 family of protein kinases are important regulators of endocytosis and actin cytoskeleton in some eukaryotic cells. In budding yeast, Prk1p phosphorylates numerous endocytic proteins including Pan1p and Sla1p. Prk1p has been observed to undergo autophosphorylation in vivo. In this study, we determined the sites and underlying role of the autophosphorylation. Two sites located in the noncatalytic region were identified to be the autophosphorylation sites. When the sites were mutated, the non-autophosphorylatable Prk1p phosphorylated Pan1p and Sla1p more efficiently than the wild-type kinase, suggesting a negative effect of the autophosphorylation. In addition, the dynamic properties of actin and the coat complex were also altered in the autophosphorylation mutant cells. Interestingly, the autophosphorylation of Prk1p was dependent on cortical localization of the kinase and could be induced by phosphorylated Sla1p. These results suggest that the autophosphorylation of Prk1p may represent a feedback mechanism possibly involved in fine-tuning the pace of progression during actin-coupled endocytosis.