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Additional file 1. Fig. S1. Seeding and staining controls for OHCS experiment. a, b) C57BL/6J OHCS injected with sterile PBS (a) or monomeric α-Syn (b) do not show any aggregation either at injection site (DG) or elsewhere at 7 dpi, as detected by conformation-specific MJF-14 (green) and pSer129 (11A5, red). Scale bars = 20 μm (a), 100 μm (b). c) Injection of PFF in α-Syn knockout OHCS does not result in any aggregate pathology at 7 dpi, due to the lack of endogenous α-Syn needed to template aggregation. Conformation-specific MJF-14 (green) was used to detect pathology. Scale bar = 100 μm. Fig. S2. Unilateral intramuscular injection of PFF elicits higher pSer129 insult in the ipsilateral lumbar DRG compared to the contralateral side. Threemonth-old TgM83+/− mice were injected with full-length (1-140) mouse α-Syn PFF (n=4) in the right hindlimb (ipsilateral, in red) or vehicle in the left hindlimb (PBS pH 7.4, contralateral, in blue). a) Proteins from lumbar DRG (L3-L5) homogenates were separated by SDS-PAGE and visualized by immunoblotting with anti-α-Syn antibody, anti-pSer129-α-Syn, anti-b-actin, and anti-b-III tubulin. b) Densitometry quantification of immunoblots as a ratio of pSer129/total α-Syn. Results are shown as mean ± SEM, as determined by one-way ANOVA followed by Tukey’s multiple comparison test. *P < 0.05. Fig. S3. Immunofluorescence detection of astrogliosis in relation to pSer129-α-Syn pathology in lumbar spinal cord, midbrain periaqueductal grey and thalamus of vehicle- injected M83 mice. a) pSer129-α-Syn(in green) co-detection with neuronal nuclei antigen (NeuN, in red) in vehicle-injected mice in dorsal (DH) and ventral (VH) and horn of lumbar spinal cord. b) pSer129-α-Syn (in green) and glial fibrillary acidic protein marker (GFAP, in red) immunoreactivity in DH and VH of lumbar spinal cord, c) midbrain periaqueductual grey (MB-PAG) and d) thalamus. DAPI (blue) was used to stain the nuclei. Scale bar = 100 μm; insets in merge show 63X magnified views. Fig. S4. Immunofluorescence quantification of astrogliosis in lumbar and ventral horn of the spinal cord, midbrain periaqueductal grey (PAG) and thalamus in vehicle and PFF- injected M83 mice. Quantification was performed on 10X views using Zen software (Zeiss). DAPI was used as a cell marker, GFAP quantitation is expressed as GFAP+ cells/mm2 in the indicated regions. Results shown as mean ± SEM as determined by ordinary one-way ANOVA followed by multiple comparison test. ***P < 0.001; ****P < 0.0001. VH, ventral horn, DH, dorsal horn, PAG, periaqueductal grey, thalamus ventroposterior. Fig. S5. Immunofluorescence for pSer129-α-Syn pathology in lumbar spinal cord of PFF- injected M83 mice. pSer129-α-Syn immunoreactivity (in green) in the dorsal (DH) and ventral (VH) horns, and the intermediate grey (IG), with 20X magnified views of grey matter (red rectangles) and white matter (yellow rectangles) as overlayimages at a) at 14 dpi and b) 21 dpi. I-X represent Rexed laminae; CC, central canal. DAPI (blue) was used to stain the nuclei.Scale bar 100 μm.

Additional file of Trans-synaptic spreading of alpha-synuclein pathology through sensory afferents leads to sensory nerve degeneration and neuropathic pain

The effect of Recombinant Botulinum Toxin A on neuropathic pain in Spared Nerve Injury mouse modelHammer RE1, Richner M2, Vaegter CB2,3, Karlsson P1,4, Nyengaard JR1,5.1. Core Center for Molecular Morphology, Section for Stereology and Microscopy, Department of Clinical Medicine, Aarhus University, Aarhus, Denmark.2. Danish Research Institute of Translational Neuroscience, Nordic EMBL Partnership for Molecular Medicine, Department of Biomedicine, Aarhus University, Aarhus, Denmark.3. The International Diabetic Neuropathy Consortium, Aarhus University Hospital, Aarhus, Denmark.4. Danish Pain Research Center, Department of Clinical Medicine, Aarhus University, Aarhus, Denmark. 5. Center for Stochastic Geometry and Advanced Bioimaging, Aarhus University, Aarhus, Denmark.Background and aims Neuropathic pain is a debilitating chronic pain disorder characterized by sensory abnormalities such as dysesthesia, hyperalgesia, and allodynia. Diabetic polyneuropathy (DPN) and traumatic nerve injury are some of the most common causes of neuropathic pain. The Db/Db model and the Spared Nerve Injury (SNI) model are validated animal models mimicking DPN and traumatic nerve injury, respectively. Published data indicate that botulinum toxin A (BoNT/A) has analgesic effects against peripheral neuropathic pain. This project aims to assess whether BoNT/A has an analgesic effect on the two mouse models representing neuropathic pain.MethodsA total of 64 mice will be used (32 control mice (C57BL/6) and 32 Db/Db mice). In each group, 16 mice will be SNI operated while the remaining 16 will be sham-operated. In each group of 16, eight will receive either vehicle or 5 pg BoNT/A pr. mouse in 20 u00b5L of gelatine phosphate buffer. Pain threshold will be assessed using Von Frey filaments and possible effects on the spinal cord, sciatic nerve, dorsal root ganglion, and brain will be evaluated using immunohistochemistry and quantitative light and electron microscopy. Results This is an ongoing study and the results will be presented at the conference. We hypothesize that rBoNT/A treatment will have an analgesic effect on both mouse models of neuropathic pain, compared with littermates that received placebo. ConclusionsIf the hypotheses hold, this project will lay the groundwork for clinical testing in human patients with neuropathic pain.

Neuropathic pain is a serious neurological disease affecting patients with peripheral nerve injuries and neuropathies. Unfortunately, the existing treatments are insufficient in giving patients pain relieve and preventing the development of neuropathic pain.Neuroinflammation is a major contributing factor in the development of neuropathic pain. It has been well accepted that activation of microglia is essential in the spinal neuroinflammatory process underlying development of neuropathic pain after peripheral nerve injury. Unpublished data from sortilin knockout mice shows that the lack of the neuronal receptor, Sortilin, decreases development of neuropathic pain after peripheral nerve injury. Because Sortilin influences the inflammatory response in macrophages, it may also affect the response in microglia and the spinal neuroinflammation. Therefore, the aim of this project is to understand how Sortilin influences the spinal microglial response following peripheral nerve injury.We plan to investigate cultures of spinal microglia from wild type and Sortilin knockout mice by comparing the cytokine profiles in culture medium, gene expression and intracellular protein levels of cytokines. Understanding the response in microglia might contribute to the knowledge of neuroinflammatory diseases in general and hopefully prove Sortilin as a potential drug target in the development of future treatments for neuropathic pain.

To investigate microdomain association of the dopamine transporter (DAT), we employed FCS (fluorescence correlation spectroscopy) and FRAP (fluorescence recovery after photobleaching). In non-neuronal cells (HEK293), FCS measurements revealed for the YFP-DAT (DAT tagged with yellow fluorescent protein) a diffusion coefficient (D) of ~3.6 × 10-9 cm2/s, consistent with a relatively freely diffusible protein. In neuronally derived cells (N2a), we were unable to perform FCS measurements on plasma membrane-associated protein due to photobleaching, suggesting partial immobilization. This was supported by FRAP measurements that revealed a lower D and a mobile fraction of the YFP-DAT in N2a cells compared to HEK293 cells. Comparison with the EGFP-EGFR (epidermal growth factor receptor) and the EGFP-beta2AR (beta2 adrenergic receptor) demonstrated that this observation was DAT specific. Both the cytoskeleton-disrupting agent cytochalasin D and the cholesterol-depleting agent methyl-beta-cyclodextrin (mbCD) increased the lateral mobility of the YFP-DAT but not that of the EGFP-EGFR. The DAT associated in part with membrane raft markers both in the N2a cells and in rat striatal synaptosomes as assessed by sucrose density gradient centrifugation. Raft association was further confirmed in the N2a cells by cholera toxin B patching. It was, moreover, observed that cholesterol depletion, and thereby membrane raft disruption, decreased both the Vmax and KM values for [3H]dopamine uptake without altering DAT surface expression. In summary, we propose that association of the DAT with lipid microdomains in the plasma membrane and/or the cytoskeleton serves to regulate both the lateral mobility of the transporter and its transport capacity

Dopaminerg neurotransmission har indflydelse på moduleringen afmotorikken, følelsesmæssig adfærd, hukommelse og bevidsthed.Derfor formodes det, at ubalancer i det dopaminerge system bidragertil sygdomsudviklingen i adskillige lidelser, deriblandt Parkinson'ssygdom, skizofreni, ADHD ("Attention Deficit Hyperactivity Disorder")og stofafhængighed.Dopamintransporteren (DAT) er et præsynaptisk protein idopaminerge nervecelle-terminaler, som afslutter dopaminergsignalering ved hurtigt at genoptage frigivet dopamin fra synapsekløften.DAT spiller derfor en vigtig rolle i vedligeholdelsen af denkorrekte dopaminerge tonus på postsynaptiske membraner og sommål for både farmaceutiske og narkotiske stoffer. Trods dennebetydning vides der meget lidt om reguleringen af transportaktivitetenog overfladetilgængeligheden af DAT i levende neuroner.DAT indeholder et C terminalt PDZ-domænebindende motiv (medsekvensen LKV), som vides at interagere med PDZ-domæneproteinerså som PICK1 ("Protein Interacting with C-Kinase 1"). I overensstemmelsemed andre rapporter har vi fundet, at ved at slette dettemotiv bliver DAT tilbageholdt i det endoplasmatiske reticulum (ER) iHEK293 og Neuro2A celler. Dette antyder, at PDZ-domæneinteraktionerspiller en kritisk rolle i DAT-targeting. Vi har dog været istand til at konstruere mutationer i DAT C terminalen, som var ude afstand til at binde PICK1, men som stadig blev udtrykt på plasmamembranen.Endvidere kunne vi konstruere andre mutationer, somstadig kunne binde PICK1, men dette var tilsyneladende ikketilstrækkeligt til at få DAT-PICK1-komplekset udtrykt påoverflademembranen. Ved progressivt at substituere med sekvens fraden β2-adrenerge receptor, og ved at indføre trippel-alaninsubstitutioner i DAT C terminalen, undersøgte vi vigtigheden afepitoper forud for LKV-sekvensen. Substitution af aminosyrerne ligeinden LKV-sekvensen med alaniner medførte tilbageholdelse af DAT iER på trods af dette konstrukts evne til fortsat at kunne binde PICK1.Dette antyder en dobbeltrolle af DAT C terminalen: En rolle i EReksport og overfladeudtryk, som er uafhængig af PDZ-interaktioner,og en stadig ikke helt afklaret rolle som involverer PDZ-interaktionermed proteiner som PICK1.viNye data foreslår en potentiel rolle for PDZ-interaktioner ireguleringen af DAT internalisering og recycling: Vi opdagede, atødelæggelse af den PDZ-domænebindende sekvens påvirkedereguleringen af DAT konstitutiv internalisering, nedbrydning og måskeogså recycling i Neuro2A celler.Vi fandt også bevis for en direkte interaktion mellem DAT og denCa2+/calmodulin-afhængige kinase II α (CaMKIIα), og denneinteraktion synes at afhænge af de samme epitoper, som eressentielle for overfladeudtryk af DAT.Endelig har vi ved hjælp af en fluorescerende kokainanalog været istand til at detektere dopamintransporteren på overfladen af levendeprimære dopaminerge neuroner. Manglen på gode ekstracellulæreantistoffer har hidtil hæmmet forsøg på at undersøge internaliseringog recycling fænomener i levende celler, men denne nye metodegiver os et unikt værktøj for at undersøge disse mekanismer i fleredetaljer.Alt i alt har de opnåede resultater givet ny indsigt i reguleringen afdopamintransporteren og vil påvirke fremtidig forskning indenfor detteområde. Dopaminergic neurotransmission is involved in the modulation oflocomotor activity, emotional behavior, memory and cognition. Hence,imbalances in the dopaminergic system in humans have beenhypothesized to contribute to the pathogenesis of a number ofillnesses, including Parkinson's disease, schizophrenia, ADHD(attention deficit hyperactivity disorder) and addiction.The dopamine transporter (DAT) is a presynaptic protein ofdopaminergic nerve terminals that terminate dopaminergic signalingby rapidly sequestering released dopamine from the synaptic cleft.The DAT therefore plays an important role in maintaining the correctdopaminergic tone on postsynaptic membranes and as a target forboth pharmaceutical and narcotic drugs. Despite this importance, littleis known about the regulation of the transport activity and surfaceavailability of DAT in the living neuron.The DAT contains a C terminal PDZ domain-binding motif (sequenceLKV) known to interact with the PDZ domain of the protein PICK1(Protein Interacting with C-kinase 1). In agreement with other reportswe found that after deletion of this motif, DAT was retained in theendoplasmic reticulum (ER) in HEK293 and Neuro2A cells,suggesting that PDZ domain interactions might be critical for DATtargeting. However, we have been able to construct mutations in theDAT C terminal that were unable to bind PICK1 but are nonethelesstargeted to the plasma membrane. Furthermore, other mutations werestill able to bind PICK1 but this was not sufficient to drive surfaceexpression of the DAT-PICK1 complex. By progressive substitutionswith β2-adrenergic receptor sequence, and by triple-alaninesubstitutions in the DAT C terminus, we examined the importance ofepitopes preceding the LKV motif. Substitution of residues justproximal to the LKV sequence with alanines caused retention of thetransporter in the ER despite preserved ability of this mutant to bindPICK1. This proposes dual roles of the DAT C terminus: a roleindependent of PDZ interactions for ER export and surface targeting,and a not fully clarified role involving PDZ interactions with proteinssuch as PICK1.New data suggest a potential role of the PDZ interaction in theregulation of DAT internalization and recycling: we found thativdisrupting the PDZ domain-binding sequence affected the regulationof constitutive internalization, degradation and potentially alsorecycling of DAT in Neuro2A cells.We also found evidence for a direct interaction between DAT and theCa2+/calmodulin dependent kinase II α (CaMKIIα) and this interactionseems to depend on the same epitopes that are essential for surfacetargeting of DAT.Finally, using a fluorescently labelled cocaine analogue we were ableto detect the dopamine transporter on the surface of living primarydopaminergic neurons. The lack of good extracellular antibodies hasso far obstructed the effort to investigate internalization and recyclingproperties in living cells, but this new method has provided us with aunique opportunity to investigate these mechanisms in further detail.All together the obtained results provide new insight into theregulation of the dopamine transporter and will direct future researchstrategies in this field.