Acta Neuropathol. 2016 Apr;131(4):621-37. doi: 10.1007/s00401-015-1512-2. Epub 2015 Dec 10.   http://www.ncbi.nlm.nih.gov/pubmed/26659577

Neuronal ceroid lipofuscinosis with DNAJC5/CSPα mutation has PPT1 pathology and exhibit aberrant protein palmitoylation.

Henderson MX1,2,3, Wirak GS1,2, Zhang YQ1,2, Dai F4, Ginsberg SD5,6, Dolzhanskaya N7, Staropoli JF8,9,10, Nijssen PC11, Lam TT12, Roth AF13, Davis NG13, Dawson G14, Velinov M15,7, Chandra SS16,17,18,


Neuronal ceroid lipofuscinoses (NCL) are a group of inherited neurodegenerative disorders with lysosomal pathology (CLN1-14). Recently, mutations in the DNAJC5/CLN4 gene, which encodes the presynaptic co-chaperone CSPα were shown to cause autosomal-dominant NCL. Although 14 NCL genes have been identified, it is unknown if they act in common disease pathways. Here we show that two disease-associated proteins, CSPα and the depalmitoylating enzyme palmitoyl-protein thioesterase 1 (PPT1/CLN1) are biochemically linked. We find that in DNAJC5/CLN4 patient brains, PPT1 is massively increased and mis-localized. Surprisingly, the specific enzymatic activity of PPT1 is dramatically reduced. Notably, we demonstrate that CSPα is depalmitoylated by PPT1 and hence its substrate. To determine the consequences of PPT1 accumulation, we compared the palmitomes from control and DNAJC5/CLN4 patient brains by quantitative proteomics. We discovered global changes in protein palmitoylation, mainly involving lysosomal and synaptic proteins. Our findings establish a functional link between two forms of NCL and serve as a springboard for investigations of NCL disease pathways.


Cysteine-string protein alpha (CSPα); Neurodegeneration; Neuronal ceroid lipofuscinosis (NCL); Palmitoyl-protein thioesterase 1 (PPT1); Palmitoylation

Delivery and Tracking of Quantum Dot Peptide Bioconjugates in an Intact Developing Avian Brain.

ACS Chemical Neurosci. Online March 2015.

Rishabh Agarwal, Miriam S. Domowicz, Nancy B. Schwartz Judy Henry, Igor Medintz, James B. Delehanty, Michael H. Stewart, Kimihiro Susumu, Alan L. Huston, Jeffrey R. Deschamps, Philip E. Dawson Valle Palomo, and Glyn Dawson

Luminescent semiconductor ∼9.5 nm nanoparticles (quantum dots: QDs) have intrinsic physiochemical and optical properties which enable us to begin to understand the mechanisms of nanoparticle mediated chemical/drug delivery. Here, we demonstrate the ability of CdSe/ZnS core/shell QDs surface functionalized with a zwitterionic compact ligand toyo brain without any apparent toxicity. Functionalized QDs were conjugated to the palmitoylated peptide WGDap-(Palmitoyl)VKIKKP9GGH6, previously shown to uniquely facilitate endosomal escape, and microinjected into the embryonic chick spinal cord canal at embryo day 4 (E4). We were subsequently able to follow the labeling of spinal cord extension into the ventricles, migratory neuroblasts, maturing brain cells, and complex structures such as the choroid plexus. QD intensity extended throughout the brain, and peaked between E8 and E11 when fluorescence was concentrated in the choroid plexus before declining to hatching (E21/P0). We observed no abnormalities in embryonic patterning or embryo survival, and mRNA in situ hybridization confirmed that, at key developmental stages, the expression pattern of genes associated with different brain cell types (brain lipid binding protein, Sox-2, proteolipid protein and Class III-β-Tubulin) all showed a normal labeling pattern and intensity. Our findings suggest that we can use chemically modified QDs to identify and track neural stem cells as they migrate, that the choroid plexus clears these injected QDs/nanoparticles from the brain after E15, and that they can deliver drugs and peptides to the developing brain.

The Role of Negative Charge in the Delivery of Quantum Dots to Neurons

Ryan Walters1, Igor L. Medintz2, James B. Delehanty2, Michael H. Stewart3, Kimihiro Susumu3, Alan L. Huston3, Philip E. Dawson4, and Glyn Dawson1,5*

Accepted for ASN Neuro April 2015.

Despite our extensive knowledge of the structure of negatively charged cell surface proteoglycans and sialoglycoconjugates in the brain, we have little understanding of how their negative charge contributes to brain function. We have previously shown that intensely photoluminescent 9 nm quantum dots (QDs) with a CdSe core, a ZnS shell, and a negatively charged compact molecular ligand coating (CL4), selectively target neurons rather than glia (Walters et al., 2012).  We now provide an explanation for this selective neuronal delivery.  In this study we compared three Zwitterionic QD coatings differing only in their regions of positive or negative charge, as well as a positively charged (NH2) polyethylene glycol (PEG) coat, for their ability to deliver the cell-membrane penetrating chaperone lipopeptide JB577 (WG(Palmitoyl)VKIKKP9G2H6) to individual cells in neonatal rat hippocampal slices.  We confirm both that preferential uptake in neurons, and the lack of uptake in glia, is strongly associated with having a region of greater negative charge on the QD coating.  Additionally, the role of negatively charged chondroitin sulfate of the extracellular matrix (ECM) in restricting uptake was further suggested by digesting neonatal rat hippocampal slices with chondroitinase ABC and showing increased uptake of QDs by oligodendrocytes.  Treatment still did not affect uptake in astrocytes or microglia.  Finally, the future potential of using QDs as vehicles for trafficking proteins into cells continues to show promise, as we show that by administering a histidine-tagged green fluorescent protein (eGFP-His6) to hippocampal slices we can observe neuronal uptake of GFP.

Quantum Dots as a Delivery Vehicle for siRNA to Cells

Getz, T., Jingdong Qin., Glyn Dawson

RNAi (RNA interference), which consists of miRNA (microRNA) and siRNA (small interfering RNA), is a potential approach to treating neurodegenerative disorders by suppression of abnormal protein activity.  Despite the inherent advantages of these small nucleic acid based drugs, there is currently no efficient or safe system for their in vivo delivery. To overcome this problem we used luminescent quantum dots (QDs) coated with zwitterionic or polyethylene glycol –derived ligands and linked to the JB577 peptide (WG (Dappal) VKIKK P9 GG His6) through the Zn coat on the Qds.  We used a gel-shift assay to show that siRNA for the smpd1 (acid  (lysosomal) sphingomyelinase), smpd3 genes (neutral sphingomyelinase), and luciferase genes bound to JB577 through the three positively charged lysine residues with an affinity similar to peptides with a Poly-Arginine sequence. Cargo delivery to cytosol was demonstrated by imaging cells by confocal microscopy.  Both QD and Cy3 labeled siRNA were visible in cell cytosol after 24-hour treatment.  Functional success was measured by comparing the QD-cargo complex with liposomal delivery systems to decrease sphingomyelinase activity.  Knockdown efficiency was also characterized by reduction Tof luciferase expression. This data suggests that QD-siRNA constructs are a robust mode of in vivo siRNA delivery and are promising a vehicles for neuro-targeted siRNA delivery.  Moreover, our QD-siRNA constructs demonstrated a significant increase in knockdown efficiency over previous reports, representing a step forward in QD mediated siRNA delivery.

Multiple sphingolipid abnormalities following cerebral microendothelial hypoxia.

Neurochem: 2014 Nov;131(4):530-40. doi: 10.1111/jnc.12836. Epub 2014 Aug 14.

Testai FD, Kilkus JP, Berdyshev E, Gorshkova I, Natarajan V, Dawson G.

Hypoxia has been previously shown to inhibit the dihydroceramide (DHC) desaturase, leading to the accumulation of DHC. In this study, we used metabolic labeling with [3H]-palmitate, HPLC/MS/MS analysis, and specific inhibitors to show numerous sphingolipid changes after oxygen deprivation in cerebral microendothelial cells. The increased DHC, particularly long-chain forms, was observed in both whole cells and detergent-resistant membranes. This was reversed by reoxygenation and blocked by the de novo sphingolipid synthesis inhibitor myriocin, but not by the neutral sphingomyelinase inhibitor GW-4869. Furthermore, oxygen deprivation of microendothelial cells increased levels of dihydro-sphingosine (DH-Sph), DH-sphingosine1-phosphate (DH-S1P), DH-sphingomyelin (DH-SM), DH-glucosylceramide (DH-GlcCer), and S1P levels. In vitro assays revealed no changes in the activity of sphingomyelinases or sphingomyelin synthase, but resulted in reduced S1P lyase activity and 40% increase in glucosylceramide synthase (GCS) activity, which was reversed by reoxygenation. Inhibition of the de novo sphingolipid pathway (myriocin) or GCS (EtPoD4) induced endothelial barrier dysfunction and increased caspase 3-mediated cell death in response to hypoxia. Our findings suggest that hypoxia induces synthesis of S1P and multiple dihydro-sphingolipids, including DHC, DH-SM, DH-GlcCer, DH-Sph and DH-S1P, which may be involved in ameliorating the effects of stroke . Progressive hypoxia leads to the accumulation of several dihydrosphingolipids in cerebral microendothelial cells. Hypoxia also increases sphingosine-1-phosphate and the activity of glucosylceramide (Glc-Cer) synthase. These changes reverse by inhibiting the de novo sphingolipid synthesis, which worsens hypoxia-induced endothelial barrier dysfunction and apoptosis, suggesting that the identified sphingolipids may be vasculoprotective.