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Chandra, S. S.

Publications and source records attributed to Chandra, S. S..

7 recordsLinked to original sources

UV induces common cutaneous amyloid-like melanosomal protein aggregates

Misfolding of aggregation-prone proteins underpins diseases known as proteinopathies. One of these proteins, alpha-synuclein, is a component of aggregates in neurodegenerative conditions such as Parkinsons disease. The melanosomal protein PMEL, which forms physiologic amyloid scaffold structures on which melanin is organized in melanosomes, similarly ectopically accumulates in the dermis in many forms of cutaneous hyperpigmentation. Here, we demonstrate in a wide range of common clinical pigmentary disorders, as well as in primary melanocyte and mouse models examined by molecular, proteomic, and electron microscopic tools, that melanocytic alpha-synuclein is a prominent component of intracellular protein aggregates bound to similar proteins as in Parkinsons disease, as well as melanized extracellular protein deposits. Using the Real Time Quaking-Induced Conversion Assay (RT-QuIC), we demonstrate that UV induces misfolded melanosomal proteins to self-propagate, augmenting this pathology in prion-like fashion. CUT&RUN chromatin profiling and single-cell RNA-seq demonstrate that melanocytes utilize microphthalmia-associated transcription factor (MITF)-regulated autophagy to counteract protein aggregation, identifying aggregate removal as a core function of tanning. In contrast to extracellular aggregation, impaired intracellular aggregate removal contributes to melanocyte senescence, which conversely exacerbates chronic hypopigmentation and photoaging-related discoloration. These findings identify melanosomal proteinopathy as a common contributor to melanocyte dysfunction and suggest aggregate-focused management approaches.

molecular biology↗

Synaptic vesicle endocytosis deficits underlie GBA-linked cognitive dysfunction in Parkinson's disease and Dementia with Lewy bodies

GBA is the major risk gene for Parkinsons disease (PD) and Dementia with Lewy Bodies (DLB), two common -synucleinopathies with cognitive deficits. We investigated the role of mutant GBA in cognitive decline by utilizing Gba (L444P) mutant, SNCA transgenic (tg), and Gba-SNCA double mutant mice. Notably, Gba mutant mice showed early cognitive deficits but lacked PD-like motor deficits or -synuclein pathology. Conversely, SNCA tg mice displayed age-related motor deficits, without cognitive abnormalities. Gba-SNCA mice exhibited both cognitive decline and exacerbated motor deficits, accompanied by greater cortical phospho--synuclein pathology, especially in layer 5 neurons. Single-nucleus RNA sequencing of the cortex uncovered synaptic vesicle (SV) endocytosis defects in excitatory neurons of Gba mutant and Gba-SNCA mice, via robust downregulation of genes regulating SV cycle and synapse assembly. Immunohistochemistry and electron microscopy validated these findings. Our results indicate that Gba mutations, while exacerbating pre-existing -synuclein aggregation and PD-like motor deficits, contribute to cognitive deficits through -synuclein-independent mechanisms, involving dysfunction in SV endocytosis.

neuroscience↗

Decoding transcriptomic signatures of Cysteine String Protein alpha-mediated synapse maintenance

Synapse maintenance is essential for generating functional circuitry and decrement in this process is a hallmark of neurodegenerative disease. While we are beginning to understand the basis of synapse formation, much less is known about synapse maintenance in vivo. Cysteine string protein (CSP), encoded by the Dnajc5 gene, is a synaptic vesicle chaperone that is necessary for synapse maintenance and linked to neurodegeneration. To investigate the transcriptional changes associated with synapse maintenance, we performed single nucleus transcriptomics on the cortex of young CSP knockout (KO) mice and littermate controls. Through differential expression and gene ontology analysis, we observed that both neurons and glial cells exhibit unique signatures in CSP KO brain. Significantly all neurons in CSP KO brains show strong signatures of repression in synaptic pathways, while upregulating autophagy related genes. Through visualization of synapses and autophagosomes by electron microscopy, we confirmed these alterations especially in inhibitory synapses. By imputing cell-cell interactions, we found that neuron-glia interactions were specifically increased in CSP KO mice. This was mediated by synaptogenic adhesion molecules, including the classical Neurexin1-Neuroligin 1 pair, suggesting that communication of glial cells with neurons is strengthened in CSP KO mice in an attempt to achieve synapse maintenance. Together, this study reveals unique cellular and molecular transcriptional changes in CSP KO cortex and provides new insights into synapse maintenance and neurodegeneration. Significance statementSynapse maintenance is important for maintaining neuronal circuitry throughout life. However, little is known about molecules that affect synapse maintenance in vivo. CSP, encoded by the Dnajc5 gene, is a synaptic vesicle chaperone that is linked to synapse maintenance and neurodegeneration. Here, we show by performing single nucleus transcriptomics of CSP KO cortex that synapse instability is related to repression in synaptic pathways and elevation of autophagy in neurons. However, we find a heterogeneity of glial responses. Additionally, interactions between neurons and glia are increased in CSP KO, mediated by synaptogenic adhesion molecules. This study provides a novel perspective on into synapse maintenance and reveals unique cellular and molecular transcriptional changes in CSP KO brains.

neuroscience↗

Dopamine transporter and synaptic vesicle sorting defects initiate auxilin-linked Parkinson's disease

Auxilin participates in the uncoating of clathrin-coated vesicles (CCVs), thereby facilitating synaptic vesicle (SV) regeneration at presynaptic sites. Auxilin (DNAJC6/PARK19) loss-of- function mutations cause early-onset Parkinsons disease (PD). Here, we utilized auxilin-knockout (KO) mice to elucidate the mechanisms through which auxilin deficiency and clathrin-uncoating deficits lead to PD. We demonstrate that auxilin KO mice display the cardinal features of PD, including progressive motor deficits, -synuclein pathology, nigral dopaminergic loss, and neuroinflammation. Through unbiased proteomic and neurochemical analyses, we demonstrate that dopamine homeostasis is disrupted in auxilin KO brains, including via slower dopamine reuptake kinetics in vivo, an effect associated with dopamine transporter misrouting into axonal membrane deformities in the dorsal striatum. We also show that elevated macroautophagy and defective SV protein sorting contribute to ineffective dopamine sequestration and homeostasis, ultimately leading to neurodegeneration. This study advances our knowledge of how presynaptic endocytosis deficits lead to dopaminergic vulnerability and pathogenesis of PD.

neuroscience↗

Identification of palmitoyl protein thioesterase 1 substrates defines roles for synaptic depalmitoylation

Loss-of-function mutations in the depalmitoylating enzyme palmitoyl protein thioesterase 1 (PPT1) cause Neuronal Ceroid Lipofuscinosis type 1 (CLN1), a devastating neurodegenerative disease. Here, we provide a resource identifying PPT1 substrates. We utilized Acyl Resin-Assisted Capture and mass spectrometry to identify proteins with increased in vivo palmitoylation in PPT1 knockout mouse brains. We then validated putative substrates through direct depalmitoylation with recombinant PPT1. This stringent screen elucidated >100 novel PPT1 substrates at the synapse, including channels and transporters, G-protein-associated molecules, endo/exocytic components, synaptic adhesion molecules, and mitochondrial proteins. Cysteine depalmitoylation sites in transmembrane PPT1 substrates frequently participate in disulfide bonds in the mature protein. We confirmed that depalmitoylation regulates disulfide bond formation in a tertiary screen analyzing post-translational modifications. Collectively, the diverse PPT1 substrates highlight the role of PPT1 in mediating synapse functions, implicate molecular pathways in the etiology of CLN1, and advance our basic understanding of the purpose of depalmitoylation. HighlightsO_LI[~]10% of palmitoylated proteins are palmitoyl protein thioesterase 1 (PPT1) substrates C_LIO_LIUnbiased proteomic approaches identify 9 distinct classes of PPT1 substrates, including synaptic adhesion molecules and endocytic proteins C_LIO_LIProtein degradation does not require depalmitoylation by PPT1 C_LIO_LIDepalmitoylation mediates disulfide bond formation in transmembrane PPT1 substrates C_LI

neuroscience↗

α-Synuclein facilitates clathrin assembly in synaptic vesicle endocytosis

-Synuclein and family members {beta}-, and {gamma}-synuclein, are presynaptic proteins that sense and generate membrane curvature, properties important for synaptic vesicle (SV) cycling. {beta}{gamma}-synuclein triple knockout (KO) neurons exhibit SV endocytosis (SVE) deficits. Here, we investigate how SVE is regulated by -synuclein. Immuno-electron microscopy (EM) of synaptosomes reveals that -synuclein relocalizes from SVs to the synaptic membrane upon stimulation, allowing -synuclein to function on presynaptic membranes during or after stimulation. On cell membranes, we observe that -synuclein is colocalized with clathrin and its adaptor AP180. Clathrin patches that contain both -synuclein and AP180 were significantly larger than clathrin patches containing either protein alone. We also find that recruitment of clathrin and AP180 recruitment to membranes are altered in the absence of synucleins. Visualizing clathrin assembly on membranes using an in vitro endocytosis reconstitution system reveals that -synuclein increases clathrin patch size and enhances clathrin lattice curvature, facilitating normal clathrin coated pit maturation. Thus, -synuclein is an endocytic accessory protein that acts at early stages of SVE to controls the size and curvature of clathrin structures on the membrane.

neuroscience↗

A Drosophila model of neuronal ceroid lipofuscinosis CLN4 reveals a hypermorphic gain of function mechanism

The autosomal dominant neuronal ceroid lipofuscinoses (NCL) CLN4 is caused by mutations in the synaptic vesicle (SV) protein CSP, which is a critical co-chaperone of Hsc70 protecting synapses from activity-dependent degeneration. We developed the first animal models of CLN4 by expressing either CLN4 mutant human CSP (hCSP) or Drosophila CSP (dCSP) in fly neurons. Similar to patients, CLN4 mutations induced excessive oligomerization of mutant hCSP and premature lethality in a dose-dependent manner. Instead of being localized to SVs, most CLN4 mutant hCSP abnormally accumulated in axons and somata, and co-localized with ubiquitinated proteins and the prelysosomal markers HRS and LAMP1. Ultrastructurally, abnormal multi-laminar membrane structures were frequently observed in axons and somata next to degenerative abnormalities. The lethality, oligomerization and prelysosomal accumulation induced by CLN4 mutations was attenuated by reducing wild type (WT) dCSP levels and enhanced by increasing WT dCSP or hCSP levels, which indicates that both CLN4 alleles resemble dominant hypermorphic gain of function mutations. Furthermore, reducing the gene dosage of Hsc70 also attenuated CLN4 phenotypes. Taken together, we suggest that CLN4 alleles resemble dominant hypermorphic gain of function mutations that drive excessive oligomerization and impair membrane trafficking.

neuroscience↗