Loss of parkin causes endoplasmic reticulum calcium dyshomeostasis by upregulation of reticulocalbin 1.

Increasing evidence suggests that astrocytes play an important role in the progression of Parkinson's disease (PD). Previous studies on our parkin knockout mouse demonstrated a higher accumulation of damaged mitochondria in astrocytes than in surrounding dopaminergic (DA) neurons, suggesting that Parkin plays a crucial role regarding their interaction during PD pathogenesis. In the current study, we examined primary mesencephalic astrocytes and neurons in a direct co-culture system and discovered that the parkin deletion causes an impaired differentiation of mesencephalic neurons. This effect required the parkin mutation in astrocytes as well as in neurons. In Valinomycin-treated parkin-deficient astrocytes, ubiquitination of Mitofusin 2 was abolished, whereas there was no significant degradation of the outer mitochondrial membrane protein Tom70. This result may explain the accumulation of damaged mitochondria in parkin-deficient astrocytes. We examined differential gene expression in the substantia nigra region of our parkin-KO mouse by RNA sequencing and identified an upregulation of the endoplasmic reticulum (ER) Ca2+ -binding protein reticulocalbin 1 (RCN1) expression, which was validated using qPCR. Immunostaining of the SN brain region revealed RCN1 expression mainly in astrocytes. Our subcellular fractionation of brain extract has shown that RCN1 is located in the ER and in mitochondria-associated membranes (MAM). Moreover, a loss of Parkin function reduced ATP-stimulated calcium-release in ER mesencephalic astrocytes that could be attenuated by siRNA-mediated RCN1 knockdown. Our results indicate that RCN1 plays an important role in ER-associated calcium dyshomeostasis caused by the loss of Parkin function in mesencephalic astrocytes, thereby highlighting the relevance of astrocyte function in PD pathomechanisms.

Differential expression of Cathepsin S and X in the spinal cord of a rat neuropathic pain model.

BACKGROUND: Ample evidence suggests a substantial contribution of cellular and molecular changes in the spinal cord to the induction and persistence of chronic neuropathic pain conditions. While for a long time, proteases were mainly considered as protein degrading enzymes, they are now receiving growing interest as signalling molecules in the pain pathology. In the present study we focused on two cathepsins, CATS and CATX, and studied their spatiotemporal expression and activity during the development and progression of neuropathic pain in the CNS of the rat 5th lumbar spinal nerve transection model (L5T). RESULTS: Immediately after the lesion, both cathepsins, CATS and CATX, were upregulated in the spinal cord. Moreover, we succeeded in measuring the activity of CATX, which was substantially increased after L5T. The differential expression of these proteins exhibited the same spatial distribution and temporal progression in the spinal cord, progressing up to the medulla oblongata in the late phase of chronic pain. The cellular distribution of CATS and CATX was, however, considerably different. CONCLUSION: The cellular distribution and the spatio-temporal development of the altered expression of CATS and CATX suggest that these proteins are important players in the spinal mechanisms involved in chronic pain induction and maintenance.

Hypoxia facilitates neurogenic dural plasma protein extravasation in mice: a novel animal model for migraine pathophysiology.

Migraine animal models generally mimic the onset of attacks and acute treatment processes. A guinea pig model used the application of meta-chlorophenylpiperazine (mCPP) to trigger immediate dural plasma protein extravasation (PPE) mediated by 5-HT2B receptors. This model has predictive value for antimigraine drugs but cannot explain the delayed onset of efficacy of 5-HT2B receptor antagonists when clinically used for migraine prophylaxis. We found that mCPP failed to induce dural PPE in mice. Considering the role 5-HT2B receptors play in hypoxia-induced pulmonary vessel muscularization, we were encouraged to keep mice under hypoxic conditions and tested whether this treatment will render them susceptible to mCPP-induced dural PPE. Following four-week of hypoxia, PPE, associated with increased transendothelial transport, was induced by mCPP. The effect was blocked by sumatriptan. Chronic application of 5-HT2B receptor or nitric oxide synthase blockers during hypoxia prevented the development of susceptibility. Here we present a migraine model that distinguishes between a migraine-like state (hypoxic mice) and normal, normoxic mice and mimics processes that are related to chronic activation of 5-HT2B receptors under hypoxia. It seems striking, that chronic endogenous activation of 5-HT2B receptors is crucial for the sensitization since 5-HT2B receptor antagonists have strong, albeit delayed migraine prophylactic efficacy.

Analgesic and antiinflammatory effects of cannabinoid receptor agonists in a rat model of neuropathic pain.

Cannabinoid receptor (CB) agonists are known to attenuate allodynia in a range of pain models, but their long-term effects and their mechanisms of action are controversial. The present study compares the antiallodynic effects of long-term treatment with a mixed CB1/CB2 (WIN55,212-2) and a selective CB2 (GW405833) cannabinoid receptor agonist and correlates these effects with their influences on spinal cord (SC) glial activation. The substances were applied daily in a rat neuropathic pain model. Tactile allodynia was assessed, and the development of gliosis was illustrated with immunohistochemical methods. Both substances reduced mechanical allodynia. Their analgesic effect was accompanied by a significant reduction in reactive gliosis and cathepsins (CAT) X and S expression. A daily injection of either substance for 8 days was sufficient to induce a sustained antiallodynic effect, which persisted up to 6 days after the last injection. The re-appearance of mechanical allodynia after this period was associated with a breakout of a strong gliotic response in the lumbar SC. Our results emphasize the therapeutic efficacy of cannabinoid receptor agonists and their inhibitory effects on the formation of gliosis.

Influence of different promoters on the expression pattern of mutated human alpha-synuclein in transgenic mice.

Two missense mutations (A53T and A30P) in the gene encoding the presynaptic protein alpha-synuclein (asyn) are associated with rare, dominantly inherited forms of Parkinson's disease (PD) and its accumulation in Lewy bodies and Lewy neurites. As an initial step in investigating the role of asyn in the pathogenesis of PD, we have generated C57BL/6 transgenic mice overexpressing the doubly mutated human asyn under the control of three different promoters; the chicken beta-actin (chbetaactin), the mouse tyrosine hydroxylase 9.6 kb (msTH) and the mouse prion protein (msprp). In this study we compared the regional and cellular expression pattern of the transgenic protein in the brain and peripheral organs of various transgenic mouse lines. Western blot analysis and immunohistochemistry consistently showed that all three promoters successfully drive the expression of the transgene. The msprp promoter was found to give the highest level of transgene expression. All promoters directed the expression into the brain and specific neuron types. However, the promoters differed with respect to (i) the expression pattern in peripheral organs, (ii) the number and (iii) the regional distribution of expressing cells in the brain. Furthermore, remarkable line-to-line variation of expression patterns was observed in mouse lines carrying the same construct. Future studies will analyze how the variations in transgene expression affect the pathogenesis in the animals.