LRRK2 regulates synaptogenesis and dopamine receptor activation through modulation of PKA activity.

Leucine-rich repeat kinase 2 (LRRK2) is enriched in the striatal projection neurons (SPNs). We found that LRRK2 negatively regulates protein kinase A (PKA) activity in the SPNs during synaptogenesis and in response to dopamine receptor Drd1 activation. LRRK2 interacted with PKA regulatory subunit IIß (PKARIIß). A lack of LRRK2 promoted the synaptic translocation of PKA and increased PKA-mediated phosphorylation of actin-disassembling enzyme cofilin and glutamate receptor GluR1, resulting in abnormal synaptogenesis and transmission in the developing SPNs. Furthermore, PKA-dependent phosphorylation of GluR1 was also aberrantly enhanced in the striatum of young and aged Lrrk2(-/-) mice after treatment with a Drd1 agonist. Notably, a Parkinson's disease-related Lrrk2 R1441C missense mutation that impaired the interaction of LRRK2 with PKARIIß also induced excessive PKA activity in the SPNs. Our findings reveal a previously unknown regulatory role for LRRK2 in PKA signaling and suggest a pathogenic mechanism of SPN dysfunction in Parkinson's disease.

Conditional expression of Parkinson's disease-related mutant α-synuclein in the midbrain dopaminergic neurons causes progressive neurodegeneration and degradation of transcription factor nuclear receptor related 1.

α-Synuclein (α-syn) plays a prominent role in the degeneration of midbrain dopaminergic (mDA) neurons in Parkinson's disease (PD). However, only a few studies on α-syn have been performed in the mDA neurons in vivo, which may be attributed to a lack of α-syn transgenic mice that develop PD-like severe degeneration of mDA neurons. To gain mechanistic insights into the α-syn-induced mDA neurodegeneration, we generated a new line of tetracycline-regulated inducible transgenic mice that overexpressed the PD-related α-syn A53T missense mutation in the mDA neurons. Here we show that the mutant mice developed profound motor disabilities and robust mDA neurodegeneration, resembling some key motor and pathological phenotypes of PD. We also systematically examined the subcellular abnormalities that appeared in the mDA neurons of mutant mice and observed a profound decrease of dopamine release, the fragmentation of Golgi apparatus, and the impairments of autophagy/lysosome degradation pathways in these neurons. To further understand the specific molecular events leading to the α-syn-dependent degeneration of mDA neurons, we found that overexpression of α-syn promoted a proteasome-dependent degradation of nuclear receptor-related 1 protein (Nurr1), whereas inhibition of Nurr1 degradation ameliorated the α-syn-induced loss of mDA neurons. Given that Nurr1 plays an essential role in maintaining the normal function and survival of mDA neurons, our studies suggest that the α-syn-mediated suppression of Nurr1 protein expression may contribute to the preferential vulnerability of mDA neurons in the pathogenesis of PD.

Astrocytic expression of Parkinson's disease-related A53T alpha-synuclein causes neurodegeneration in mice.

BACKGROUND: Parkinson's disease (PD) is the most common movement disorder. While neuronal deposition of alpha-synuclein serves as a pathological hallmark of PD and Dementia with Lewy Bodies, alpha-synuclein-positive protein aggregates are also present in astrocytes. The pathological consequence of astrocytic accumulation of alpha-synuclein, however, is unclear. RESULTS: Here we show that PD-related A53T mutant alpha-synuclein, when selectively expressed in astrocytes, induced rapidly progressed paralysis in mice. Increasing accumulation of alpha-synuclein aggregates was found in presymptomatic and symptomatic mouse brains and correlated with the expansion of reactive astrogliosis. The normal function of astrocytes was compromised as evidenced by cerebral microhemorrhage and down-regulation of astrocytic glutamate transporters, which also led to increased inflammatory responses and microglial activation. Interestingly, the activation of microglia was mainly detected in the midbrain, brainstem and spinal cord, where a significant loss of dopaminergic and motor neurons was observed. Consistent with the activation of microglia, the expression level of cyclooxygenase 1 (COX-1) was significantly up-regulated in the brain of symptomatic mice and in cultured microglia treated with conditioned medium derived from astrocytes over-expressing A53T alpha-synuclein. Consequently, the suppression of COX-1 activities extended the survival of mutant mice, suggesting that excess inflammatory responses elicited by reactive astrocytes may contribute to the degeneration of neurons. CONCLUSIONS: Our findings demonstrate a critical involvement of astrocytic alpha-synuclein in initiating the non-cell autonomous killing of neurons, suggesting the viability of reactive astrocytes and microglia as potential therapeutic targets for PD and other neurodegenerative diseases.

Phosphorylation of ezrin/radixin/moesin proteins by LRRK2 promotes the rearrangement of actin cytoskeleton in neuronal morphogenesis.

Leucine-rich repeat kinase 2 (LRRK2) functions as a putative protein kinase of ezrin, radixin, and moesin (ERM) family proteins. A Parkinson's disease-related G2019S substitution in the kinase domain of LRRK2 further enhances the phosphorylation of ERM proteins. The phosphorylated ERM (pERM) proteins are restricted to the filopodia of growing neurites in which they tether filamentous actin (F-actin) to the cytoplasmic membrane and regulate the dynamics of filopodia protrusion. Here, we show that, in cultured neurons derived from LRRK2 G2019S transgenic mice, the number of pERM-positive and F-actin-enriched filopodia was significantly increased, and this correlates with the retardation of neurite outgrowth. Conversely, deletion of LRRK2, which lowered the pERM and F-actin contents in filopodia, promoted neurite outgrowth. Furthermore, inhibition of ERM phosphorylation or actin polymerization rescued the G2019S-dependent neuronal growth defects. These data support a model in which the G2019S mutation of LRRK2 causes a gain-of-function effect that perturbs the homeostasis of pERM and F-actin in sprouting neurites critical for neuronal morphogenesis.

Leucine-rich repeat kinase 2 regulates the progression of neuropathology induced by Parkinson's-disease-related mutant alpha-synuclein.

Mutations in alpha-synuclein and Leucine-rich repeat kinase 2 (LRRK2) are linked to autosomal dominant forms of Parkinson's disease (PD). However, little is known about any potential pathophysiological interplay between these two PD-related genes. Here we show in transgenic mice that although overexpression of LRRK2 alone did not cause neurodegeneration, the presence of excess LRRK2 greatly accelerated the progression of neuropathological abnormalities developed in PD-related A53T alpha-synuclein transgenic mice. Moreover, we found that LRRK2 promoted the abnormal aggregation and somatic accumulation of alpha-synuclein in A53T mice, which likely resulted from the impairment of microtubule dynamics, Golgi organization, and the ubiquitin-proteasome pathway. Conversely, genetic ablation of LRRK2 preserved the Golgi structure and suppressed the aggregation and somatic accumulation of alpha-synuclein, and thereby delayed the progression of neuropathology in A53T mice. These findings demonstrate that overexpression of LRRK2 enhances alpha-synuclein-mediated cytotoxicity and suggest inhibition of LRRK2 expression as a potential therapeutic option for ameliorating alpha-synuclein-induced neurodegeneration.

The neural pathway of galanin in the hypothalamic arcuate nucleus of rats: activation of beta-endorphinergic neurons projecting to periaqueductal gray matter.

We have previously shown that microinjection of galanin into the arcuate nucleus of hypothalamus (ARC) produced antinociceptive effects in rats (Sun et al., 2003a). In this study, the neural pathway of galanin from ARC to midbrain periaqueductal gray (PAG) in nociceptive modulation was investigated. The hindpaw withdrawal latencies (HWLs) with noxious thermal and mechanical stimulation were assessed by the hotplate and the Randall Selitto tests. Intra-ARC administration of 0.1, 0.5, or 1 nmol of galanin induced significant increases in HWLs of rats. The galanin-induced increases in HWLs were inhibited by injection of 10 microg of the opioid receptor antagonist naloxone or 1 nmol of the mu-opioid receptor antagonist beta-funaltrexamine (beta-FNA) into PAG, suggesting that the antinociceptive effects induced by intra-ARC injection of galanin occur via the neural pathway from ARC to PAG. Furthermore, our results demonstrate that the galaninergic fibers directly innervated the beta-endorphinergic neurons in ARC by immunofluorescent methods. Taken together, our results suggest that galanin produces antinociceptive effects in the ARC of rats by activating the beta-endorphinergic pathway from ARC to PAG.

Involvement of galanin in nociceptive regulation in the arcuate nucleus of hypothalamus in rats with mononeuropathy.

The hindpaw withdrawal latencies (HWLs) to noxious thermal and mechanical stimulation increased significantly after intra-hypothalamic arcuate nucleus (ARC) injection of galanin in mononeuropathic rats, while intra-ARC injection of the putative antagonist of galanin receptors markedly reduced the HWLs. The number of galaninergic neurons in the ARC increased in rats with mononeuropathy than that in normal rats. The results demonstrated that both endogenous and exogenous galanin were involved in the regulation of nociception in the ARC of rats with peripheral nerve injury.

The colocalization of CGRP receptor and AMPA receptor in the spinal dorsal horn neuron of rat: a morphological and electrophysiological study.

Both the calcitonin gene-related peptide (CGRP) receptor and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor are involved in the transmission of sensory information from primary afferent to the spinal cord. The present study found that there was a colocalization of CGRP receptor and AMPA receptor in a single spinal dorsal horn neuron in rat determined by double immunofluorescence labeling image methods. Furthermore, our results showed that the evoked discharge frequency of the wide dynamic range (WDR) neuron, one type of the dorsal horn neurons, increased significantly after micro-iontophoretic delivery of CGRP or AMPA alone tested by extracellular recording, indicating a functional colocalization of CGRP receptor and AMPA receptor in a single spinal dorsal horn neuron. The results of the present study found a morphological and functional colocalization of the CGRP receptor and AMPA receptor in a single dorsal horn neuron that involved in the transmission and modulation of sensory information from primary afferent to the spinal cord in rats.

Involvement of opioid receptors in oxytocin-induced antinociception in the nucleus accumbens of rats.

UNLABELLED: Antinociceptive effects of oxytocin have been demonstrated in mice, rats, dogs, and humans. It has been shown that oxytocin receptors and fibers with oxytocin were distributed in the nucleus accumbens (NAc) of rats. The present study was performed to investigate the regulating role of oxytocin in nociception in the NAc of rats. Intra-NAc administration of oxytocin-induced dose-dependent increases in the hindpaw withdrawal latency (HWL) to noxious thermal and mechanical stimulation in rats, indicating that oxytocin has antinociceptive effects in the NAc of rats. Furthermore, the oxytocin-induced antinociceptive effects were attenuated by intra-NAc administration of the opioid-receptor antagonist naloxone, suggesting that the endogenous opioid system is involved in the oxytocin-induced antinociception in the NAc. Moreover, the oxytocin-induced antinociception was attenuated by intra-NAc injection of the kappa-receptor antagonist nor-binaltorphimine (nor-BNI) and the mu-receptor antagonist beta-funaltrexamine, but not by the delta-receptor antagonist naltrindole, demonstrating the involvements of mu- and kappa-receptors, but not delta-receptor, in the oxytocin-induced antinociception in the NAc of rats. PERSPECTIVE: This article supplements the evidence that oxytocin regulates nociception in the central nervous system. It presents additional material for clinical application of oxytocin as an analgesia drug.

An antinociceptive role of galanin in the arcuate nucleus of hypothalamus in intact rats and rats with inflammation.

In the arcuate nucleus of hypothalamus (ARC), galaninergic fibers form synaptic contacts with proopiomelanocortin neurons, which are involved in pain modulation. The present study assessed the role of exogenous and endogenous galanin in the modulation of nociception in the ARC of rats. The hindpaw withdrawal latency (HWL) to thermal and mechanical stimulation was assessed by the hot-plate test and the Randall Selitto Test. Intra-ARC injection of galanin dose-dependently increased the HWLs in intact rats, indicating an antinociceptive role of exogenous galanin in the ARC. The antinociceptive effect of galanin was blocked by following intra-ARC injection of galantide, a putative galanin receptor antagonist, suggesting that the antinociceptive effect of galanin is mediated by galanin receptors. Moreover, intra-ARC injection of galanin increased the HWL in rats with inflammation. Intra-ARC administration of galantide alone reduced the HWLs in rats with inflammation, while there were no influences of galantide on the HWL in intact rats. Taken together, the results show that galanin has an antinociceptive role in the ARC of intact rats and rats with inflammation.