Biology subjects

Pan, P.-Y.

Publications and source records attributed to Pan, P.-Y..

5 recordsLinked to original sources

bioRxiv2022

Synaptojanin1 regulates lysosomal functions in ventral midbrain neurons

A major pathological hallmark of Parkinsons Disease (PD) is the manifestation of Lewy bodies comprised of alpha-synuclein (-syn). The accumulation of -syn enriched protein aggregates is thought to arise from dysfunction in degradation systems within the brain. Recently, missense mutations of SYNJ1 encoding the SAC1 and 5-phosphatase domains have been found in families with hereditary early-onset Parkinsonism. Previous studies showed that Synj1 haploinsufficiency (Synj1+/-) leads to PD-like behavioral and pathological changes in mice, including the accumulation of the autophagy substrate p62 and pathological -syn proteins in the midbrain (MB) and striatum. In this study, we aim to investigate the neuronal degradation pathway using the Synj1+/- MB culture as a model. Our data suggests that autophagy flux and cumulative autophagosome formation is unaltered at baseline in Synj1+/- MB neurons. However, lysosome number is reduced with a similar decrease in lysosomal proteins, including LAMP1, LAMP2, and LAMP2A. Lysosomes are hyperacidified with enhanced enzymatic activity in Synj1+/- MB neurons. Using a combination of light and electron microscopy, we show that lysosomal changes are primarily associated with a lack of SAC1 activity. Consistently, expressing the SYNJ1 R258Q mutant in N2a cells reduces the lysosome number. Interestingly, the lysosomal defects in Synj1+/- neurons does not impact the clearance of exogenously expressed wild-type -syn; however, the clearance of -syn A53T was impaired in the axons of Synj1+/- MB neurons. Taken together, our results suggest axonal vulnerability to lysosomal defects in Synj1 deficient MB neurons. Significance StatementIn the study, Zhu et al. discovered a previously uncharacterized role of Synj1 in regulating lysosomal number, protein, and acidity in ventral midbrain neurons. These alterations are associated with a specific impairment in the clearance of -syn A53T, but not WT -syn in axons, suggesting an essential role of Synj1 in axonal degradative capacity under pathological stress. This work in cultured mammalian neurons complements recent research efforts in Drosophila, C. elegans and zebra fish, and provides a novel insight for the role Synj1 in neuronal autolysosomal function.

neuroscience↗

bioRxiv2022

A novel pH-sensitive reporter reveals the cocaine-regulated trafficking of dopamine transporters in neuronal processes

The dopamine transporter (DAT) mediated DA reuptake is a major molecular mechanism for termination of dopaminergic signaling in the brain. Psychoactive substances such as cocaine act by inhibition of plasma membrane DAT function as well as by altering its expression. The precise manner and mechanism by which cocaine regulates DAT trafficking, especially at neuronal processes, are poorly understood. We have now engineered a novel pH-sensitive reporter for DAT by conjugating pHluorin to the second exofacial loop of human DAT. We show that DAT-pHluorin can be used to study DAT localization and its dynamic trafficking at neuronal processes. Using DAT-pHluorin we show that unlike neuronal soma and dendrites, which contain majority of the DATs in weakly acidic intracellular compartments, axonal DATs at both shafts and boutons are primarily (75%) localized to the plasma membrane, while varicosities contain abundant intracellular DAT within acidic intracellular structures. Using this novel reporter, we show, for the first time, that cocaine exposure leads to a brief DAT internalization followed by membrane reinsertion that lasts for days. We further show that the cocaine-induced DAT trafficking is sensitive to the activities of Synaptojanin1 phosphatase. Thus, our study using the newly engineered DAT optical reporter reveals the previously unknown dynamics and molecular regulation for cocaine-regulated DAT trafficking in neuronal processes.

neuroscience↗

bioRxiv2021

Chemoproteomic profiling reveals cellular targets of nitro-fatty acids

Nitro-fatty acids are a class of endogenous electrophilic lipid mediators with anti-inflammatory and cytoprotective effects in a wide range of inflammatory and fibrotic disease models. While these beneficial biological effects of nitro-fatty acids are mainly attributed to their ability to form covalent adducts with proteins, only a small number of proteins are known to be nitro-alkylated and the scope of protein nitro-alkylation remains undetermined. Here we describe the synthesis and application of a clickable nitro-fatty acid probe for the detection and first global identification of mammalian proteins that are susceptible to nitro-alkylation. 184 high confidence nitro-alkylated proteins were identified in human macrophages, majority of which are novel targets of nitro-fatty acids, including Extended synaptotagmin 2 (ESYT2), Signal transducer and activator of transcription 3 (STAT3), Toll-like receptor 2 (TLR2), Retinoid X receptor alpha (RXR) and Glucocorticoid receptor (NR3C1). In particular, we showed that 9-nitro-oleate covalently modified and inhibited dexamethasone binding to NR3C1. Bioinformatic analyses revealed that nitro-alkylated proteins are highly enriched in endoplasmic reticulum and transmembrane proteins, and are overrepresented in lipid metabolism and transport pathways. This study significantly expands the scope of protein substrates targeted by nitro-fatty acids in living cells and provides a useful resource towards understanding the pleiotropic biological roles of nitro-fatty acids as signaling molecules or as multi-target therapeutic agents.

biochemistry↗

bioRxiv2021

Synaptojanin1 deficiency upregulates basal level autophagosome formation in astrocytes

Macroautophagy (hereafter, autophagy) dysregulation is implicated in multiple neurological disorders. While the autophagy pathways are heavily investigated in heterologous cells and neurons, how autophagy is regulated in the astrocyte, the most abundant cell type in the mammalian brain, is less understood. Here we report that Synaptojanin1 (Synj1), a neuron enriched lipid phosphatase, is expressed in low levels in astrocytes and represses autophagy at the basal level. Synj1 is encoded by the Synj1 gene, whose missense mutations are linked to Parkinsonism with seizure. While the best-known role of Synj1 is to facilitate synaptic vesicle recycling, recent studies suggest that Synj1 also regulates autophagy. Our previous study using the Synj1 haploinsufficient (Synj1+/-) mouse demonstrated that Synj1 deficiency was associated with an age-dependent autophagy impairment in multiple brain regions. We now use cultured astrocytes from Synj1 deficient mice to investigate its role in astrocyte autophagy. We demonstrate that Synj1 deficient astrocytes exhibit increased LC3 puncta, which is more pronounced when lysosomal acidification is blocked. The increased autophagosome formation is accompanied by reduced autophagy substrate, p62, but an insensitivity to starvation induced autophagy clearance. Moreover, we show, for the first time, that the Parkinsonism associated R839C mutation impacts astrocyte autophagy. The profound impact of this mutation on Synj1s phosphatase functions results in elevated basal level autophagosome formation and clearance that mimics Synj1 deletion. We find that energy sensing molecules, including mTOR and AMPK, are altered in Synj1 deficient astrocytes, which may contribute to the enhanced basal level autophagy.

cell biology↗

bioRxiv2017

Haploinsufficiency of Parkinsonism Gene SYNJ1 Contributes to Dopamine neuron Vulnerability in Aged Mice

Parkinsons disease (PD) is an age-dependent neurodegenerative disorder characterized by the loss of substantia nigra dopaminergic (DAergic) neurons in ventral midbrain (MB). Identification of interactions between aging and the known risk variants is crucial to understanding the etiology of PD. Recessive mutations in SYNJ1 have recently been linked to familial early-onset atypical Parkinsonism. We now show an age-dependent decline of SYNJ1 expression in the striatum as well as in striatal DAergic terminals of aged mice. Heterozygous deletion of SYNJ1 in mice causes selective elevation of PIP2 in the MB, and manipulation of PIP2 levels also impairs synaptic vesicle recycling preferentially in MB neurons. SYNJ1+/- mice display progressive PD-like behavioral alterations and DAergic terminal degeneration. Furthermore, we found down-regulation of human SYNJ1 transcripts in a subset of sporadic PD brains, corroborating the role of an age-dependent decrease in SYNJ1 in predisposing DAergic neuron vulnerability and PD pathogenesis.

neuroscience↗