LRRK2 secretion in exosomes is regulated by 14-3-3

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset Parkinson's disease (PD). Emerging evidence suggests a role for LRRK2 in the endocytic pathway. Here, we show that LRRK2 is released in extracellular microvesicles (i.e. exosomes) from cells that natively express LRRK2. LRRK2 localizes to collecting duct epithelial cells in the kidney that actively secrete exosomes into urine. Purified urinary exosomes contain LRRK2 protein that is both dimerized and phosphorylated. We provide a quantitative proteomic profile of 1673 proteins in urinary exosomes and find that known LRRK2 interactors including 14-3-3 are some of the most abundant exosome proteins. Disruption of the 14-3-3 LRRK2 interaction with a 14-3-3 inhibitor or through acute LRRK2 kinase inhibition potently blocks LRRK2 release in exosomes, but familial mutations in LRRK2 had no effect on secretion. LRRK2 levels were overall comparable but highly variable in urinary exosomes derived from PD cases and age-matched controls, although very high LRRK2 levels were detected in some PD affected cases. We further characterized LRRK2 exosome release in neurons and macrophages in culture, and found that LRRK2-positive exosomes circulate in cerebral spinal fluid (CSF). Together, these results define a pathway for LRRK2 extracellular release, clarify one function of the LRRK2 14-3-3 interaction and provide a foundation for utilization of LRRK2 as a biomarker in clinical trial

LRRK2 secretion in exosomes is regulated by 14-3-3

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset Parkinson's disease (PD). Emerging evidence suggests a role for LRRK2 in the endocytic pathway. Here, we show that LRRK2 is released in extracellular microvesicles (i.e. exosomes) from cells that natively express LRRK2. LRRK2 localizes to collecting duct epithelial cells in the kidney that actively secrete exosomes into urine. Purified urinary exosomes contain LRRK2 protein that is both dimerized and phosphorylated. We provide a quantitative proteomic profile of 1673 proteins in urinary exosomes and find that known LRRK2 interactors including 14-3-3 are some of the most abundant exosome proteins. Disruption of the 14-3-3 LRRK2 interaction with a 14-3-3 inhibitor or through acute LRRK2 kinase inhibition potently blocks LRRK2 release in exosomes, but familial mutations in LRRK2 had no effect on secretion. LRRK2 levels were overall comparable but highly variable in urinary exosomes derived from PD cases and age-matched controls, although very high LRRK2 levels were detected in some PD affected cases. We further characterized LRRK2 exosome release in neurons and macrophages in culture, and found that LRRK2-positive exosomes circulate in cerebral spinal fluid (CSF). Together, these results define a pathway for LRRK2 extracellular release, clarify one function of the LRRK2 14-3-3 interaction and provide a foundation for utilization of LRRK2 as a biomarker in clinical trials

Convergence of genetic and environmental factors on parvalbumin-positive interneurons in schizophrenia

Schizophrenia etiology is thought to involve an interaction between genetic and environmental factors during postnatal brain development. However, there is a fundamental gap in our understanding of the molecular mechanisms by which environmental factors interact with genetic susceptibility to trigger symptom onset and disease progression. In this review, we summarize the most recent findings implicating oxidative stress as one mechanism by which environmental insults, especially early life social stress, impact the development of schizophrenia. Based on a review of the literature and the results of our own animal model, we suggest that environmental stressors such as social isolation render parvalbumin-positive interneurons vulnerable to oxidative stress. We previously reported that social isolation stress exacerbates many of the schizophrenia-like phenotypes seen in a conditional genetic mouse model of schizophrenia in which NMDARs are selectively ablated in half of cortical and hippocampal interneurons during early postnatal development (Belforte et al., 2010). We have since revealed that this social isolation-induced effect is caused by impairments in the antioxidant defense capacity in the parvalbumin-positive interneurons in which NMDARs are ablated. We propose that this effect is mediated by the down-regulation of PGC-1α, a master regulator of mitochondrial energy metabolism and anti-oxidant defense, following the deletion of NMDARs (Jiang et al, 2013). Other potential molecular mechanisms underlying redox dysfunction upon gene and environmental interaction will be discussed, with a focus on the unique properties of parvalbumin-positive interneurons

The NRF2-Dependent Transcriptional Regulation of Antioxidant Defense Pathways: Relevance for Cell Type-Specific Vulnerability to Neurodegeneration and Therapeutic Intervention

Oxidative stress has been implicated in the etiology and pathobiology of various neurodegenerative diseases. At baseline, the cells of the nervous system have the capability to regulate the genes for antioxidant defenses by engaging nuclear factor erythroid 2 (NFE2/NRF)-dependent transcriptional mechanisms, and a number of strategies have been proposed to activate these pathways to promote neuroprotection. Here, we briefly review the biology of the transcription factors of the NFE2/NRF family in the brain and provide evidence for the differential cellular localization of NFE2/NRF family members in the cells of the nervous system. We then discuss these findings in the context of the oxidative stress observed in two neurodegenerative diseases, Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS), and present current strategies for activating NFE2/NRF-dependent transcription. Based on the expression of the NFE2/NRF family members in restricted populations of neurons and glia, we propose that, when designing strategies to engage these pathways for neuroprotection, the relative contributions of neuronal and non-neuronal cell types to the overall oxidative state of tissue should be considered, as well as the cell types which have the greatest intrinsic capacity for producing antioxidant enzymes

Dysregulation of PGC-1α-Dependent Transcriptional Programs in Neurological and Developmental Disorders: Therapeutic Challenges and Opportunities

Substantial evidence indicates that mitochondrial impairment contributes to neuronal dysfunction and vulnerability in disease states, leading investigators to propose that the enhancement of mitochondrial function should be considered a strategy for neuroprotection. However, multiple attempts to improve mitochondrial function have failed to impact disease progression, suggesting that the biology underlying the normal regulation of mitochondrial pathways in neurons, and its dysfunction in disease, is more complex than initially thought. Here, we present the proteins and associated pathways involved in the transcriptional regulation of nuclear-encoded genes for mitochondrial function, with a focus on the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1α). We highlight PGC-1α’s roles in neuronal and non-neuronal cell types and discuss evidence for the dysregulation of PGC-1α-dependent pathways in Huntington’s Disease, Parkinson’s Disease, and developmental disorders, emphasizing the relationship between disease-specific cellular vulnerability and cell-type-specific patterns of PGC-1α expression. Finally, we discuss the challenges inherent to therapeutic targeting of PGC-1α-related transcriptional programs, considering the roles for neuron-enriched transcriptional coactivators in co-regulating mitochondrial and synaptic genes. This information will provide novel insights into the unique aspects of transcriptional regulation of mitochondrial function in neurons and the opportunities for therapeutic targeting of transcriptional pathways for neuroprotection

Transcriptional profile of general striatal projection markers.

<p>Gene expression of Gad67 (Gad1), calbindin (Calb1), and μ opioid receptor (Oprm) was measured in striatum of PGC-1α and R6/2 HD mice and littermate controls at four and twelve weeks using q-RT-PCR. No changes were found in mice lacking PGC-1α (<b>A</b>), but the striosome marker Oprm was significantly increased in R6/2 compared to WT mice (<b>B</b>) at four weeks of age. By twelve weeks of age, the matrix marker Calb1 and the striosome marker Oprm were significantly increased in PGC-1α ^+/− and ^−/− compared to ^+/+ mice (<b>C</b>), and Gad1 was significantly decreased in R6/2 HD mice (<b>D</b>). For A and C, one-way ANOVA followed by Fisher's LSD. For B and D, two-tailed t-tests. *p<0.05. **p<0.005. n/group indicated on histograms. Data presented as mean ± SEM.</p

Transcriptional profile of indirect pathway markers.

<p>Gene expression of preproenkephalin (Penk1) and dopamine receptor 2 (Drd2) was measured in striatum of PGC-1α and R6/2 HD mice and littermate controls at four and twelve weeks using q-RT-PCR. <b>A.</b> At four weeks of age, expression of Penk1 and Drd2 was significantly decreased in PGC-1α ^−/− compared to ^+/+ and ^+/− mice. <b>B.</b> No changes were observed in R6/2 HD mice at this age. <b>C.</b> By twelve weeks of age, Penk1 was significantly increased in PGC-1α ^−/− and ^+/− compared to ^+/+ mice, and Drd2 was significantly increased in PGC-1α ^−/− compared to ^+/+ and ^+/− mice. <b>D.</b> Both Penk1 and Drd2 were significantly decreased in R6/2 HD mice at this age. For A and C, one-way ANOVA followed by Fisher's LSD. For B and D, two-tailed t-tests. *p<0.05. **p<0.005. ***p<0.0005. n/group indicated on histograms. Data presented as mean ± SEM.</p

Early postnatal development of spongiform vacuolation in PGC-1α ^−/− striatum.

<p>Hematoxylin and eosin (H&E) stained coronal sections from PGC-1<b>α ^−/−</b> mice at two, four, and twelve weeks demonstrate the appearance of vacuoles between two weeks (<b>A</b>; no vacuoles present) and four weeks (<b>B</b>; arrowheads) of age. Notably, vacuoles do not appear to increase in size or number between four weeks and twelve weeks (<b>C</b>) of age, indicating that neurodegeneration does not progress as animals transition from the early postnatal period to adulthood. No vacuoles were observed in age-matched PGC-1α <b>^+/+</b> littermates (data not shown). A′–C′ are higher power images from the sections in A–C. Cx, cortex. Cc, corpus callosum. Str, striatum. Scale bars = 250 µm.</p