Mutations in ␣-synuclein (␣S) and parkin cause heritable forms of Parkinson disease (PD). We hypothesized that neuronal parkin, a known E3 ubiquitin ligase, facilitates the formation of Lewy bodies (LBs), a pathological hallmark of PD. Here, we report that affinity-purified parkin antibodies labeled classical LBs in substantia nigra sections from four related human disorders: sporadic PD, inherited ␣S-linked PD, dementia with LBs (DLB), and LB-positive, parkin-linked PD. Anti-parkin antibodies also detected LBs in entorhinal and cingulate cortices from DLB brain and ␣S inclusions in sympathetic gangliocytes from sporadic PD. Double labeling with confocal microscopy of DLB midbrain sections revealed that ϳ90% of anti-␣S-reactive LBs were also detected by a parkin antibody to amino acids 342 to 353. Accordingly, parkin proteins, including the 53-kd mature isoform, were present in affinity-isolated LBs from DLB cortex. Fluorescence resonance energy transfer and immunoelectron microscopy showed that ␣S and parkin co-localized within brainstem and cortical LBs. Biochemically, parkin appeared most enriched in cytosolic and postsynaptic fractions of adult rat brain, but also in purified, ␣S-rich presynaptic elements that additionally contained parkin's E2-binding partner, UbcH7. We conclude that parkin and UbcH7 are present with ␣S in subcellular compartments of normal brain and that parkin frequently co-localizes with ␣S aggregates in the characteristic LB inclusions of PD and DLB. These results suggest that functional parkin proteins may be required during LB formation. 3,4 Clinically, a subset of parkin-linked cases are virtually indistinguishable from sporadic PD. 4,5 Neuropathologically, sporadic and ␣S-linked PD share neuronal loss and Lewy body (LB) inclusions in selective brainstem nuclei.6 -8 The etiology of sporadic PD remains largely unknown, but mechanisms involving oxidative stress and mitochondrial dysfunction have been implicated. 9 In the related disorder, dementia with LBs (DLB), both brainstem and cortical neurons are affected by LB formation.10 -12 ␣S and ubiquitin (Ub) represent the principal known protein constituents of these inclusions. [13][14][15] In contrast, LBs are generally absent in parkin-linked PD brains, 16 -20 with one recently reported exception. 21The 465-amino acid parkin protein contains an N-terminal Ub-like domain linked to a C-terminal RING box 22 that contains two canonical RING-finger domains (C 3 H 1 C 4 ) and an imperfect, third RING finger motif (C 6 H 1 C 1 ), also referred to as "in-between-RING" 23 or "double-RING-finger-linked" domain 24 ( Figure 1A). As such, parkin is a likely member of a class of zinc-binding proteins that comprises several Ub ligases. 25,26 Indeed, parkin has been shown to act as an E3 Ub ligase in transfected cell cultures and in vitro assays, in which it principally recruits one of two E2 Ub-conjugating proteins at its RING box, UbcH7 22,27,28 or UbcH8. 29 In general, the transfer of more than four activated Ub molecules from an E2 protein onto a...

δ-Catenin is a neuronal protein that contains 10 Armadillo motifs and binds to the juxtamembrane segment of classical cadherins. We report that δ-catenin interacts with cortactin in a tyrosine phosphorylation–dependent manner. This interaction occurs within a region of the δ-catenin sequence that is also essential for the neurite elongation effects. Src family kinases can phosphorylate δ-catenin and bind to δ-catenin through its polyproline tract. Under conditions when tyrosine phosphorylation is reduced, δ-catenin binds to cortactin and cells extend unbranched primary processes. Conversely, increasing tyrosine phosphorylation disrupts the δ-catenin–cortactin complex. When RhoA is inhibited, δ-catenin enhances the effects of Rho inhibition on branching. We conclude that δ-catenin contributes to setting a balance between neurite elongation and branching in the elaboration of a complex dendritic tree.

␦-Catenin is a synaptic adherens junction protein pivotally positioned to serve as a signaling sensor and integrator. Expression of ␦-catenin induces filopodia-like protrusions in neurons. Here we show that the small GTPases of the Rho family act coordinately as downstream effectors of ␦-catenin. A dominant negative Rac prevented ␦-catenin-induced protrusions, and Cdc42 activity was dramatically increased by ␦-catenin expression. A kinase dead LIMK (LIM kinase) and a mutant Cofilin also prevented ␦-catenin-induced protrusions. To link the effects of ␦-catenin to a physiological pathway, we noted that (S)-3,5-dihydroxyphenylglycine (DHPG) activation of metabotropic glutamate receptors induced dendritic protrusions that are very similar to those induced by ␦-catenin. Furthermore, ␦-catenin RNA-mediated interference can block the induction of dendritic protrusions by DHPG. Interestingly, DHPG dissociated PSD-95 and N-cadherin from the ␦-catenin complex, increased the association of ␦-catenin with Cortactin, and induced the phosphorylation of ␦-catenin within the sites that bind to these protein partners.␦-Catenin is a component of the synaptic adherens junction that is necessary for normal learning and memory (1). In the absence of ␦-catenin, mice have severe deficits in several types of memory as well as synaptic plasticity. However, the functional basis for these deficits is not obvious, particularly because the morphological changes in ␦-catenin null mice are minimal. ␦-Catenin contains 10 Armadillo repeats (a 42-amino acid motif, originally described in the Drosophila segment polarity gene, armadillo) spaced in the characteristic arrangement of all members of this gene family which includes the prototypical member, p120 ctn , as well as p0071, ARVCF (Armadillo Repeat gene deleted in Velo-Cardio-Facial syndrome) (2), and the plakophilins, both components of the desmosome (3-6). The core functions of this protein family are stabilization of cadherins by binding to a highly conserved sequence in the juxtamembrane region and regulatory coordination over Rho GTPases (7). ␦-Catenin is localized to the post-synaptic adherens junction, collaborates with Rho GTPases to set a balance between neurite elongation and branching, and robustly induces dendritic protrusions (8). Among the cadherin binding family members, ␦-catenin is the only one that is a neural-specific protein. However, ␦-catenin null mice develop normally, whereas p120 ctn can regulate synapse and spine development (9).Because both p120 ctn and ␦-catenin are expressed in neurons, an important question is the added functionality provided by co-expression of these paralogs. ⌱n contrast to p120 ctn , ␦-catenin contains a short carboxyl-terminal motif that corresponds to a ligand sequence for PDZ (postsynaptic density-95 (PSD-95) 6 /discs large/zona occludens-1) domain-containing proteins. Through the versatility of this domain, the multiple complex interactions of ␦-catenin with the synapse arise. ␦-Catenin binds to the synaptic scaffolding molecule (S-SCAM) (...

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ABSTRACT:Transcriptional profiling (TP) offers a powerful approach to identify genes activated during memory formation and, by inference, the molecular pathways involved. Trace eyeblink conditioning is well suited for the study of regional gene expression because it requires the hippocampus, whereas the highly parallel task, delay conditioning, does not. First, we determined when gene expression was most regulated during trace conditioning. Rats were exposed to 200 trials per day of paired and unpaired stimuli each day for 4 days. Changes in gene expression were most apparent 24 h after exposure to 200 trials. Therefore, we profiled gene expression in the hippocampus 24 h after 200 trials of trace eyeblink conditioning, on multiple arrays using additional animals. Of 1,186 genes on the filter array, seven genes met the statistical criteria and were also validated by real-time polymerase chain reaction. These genes were growth hormone (GH), c-kit receptor tyrosine kinase (c-kit), glutamate receptor, metabotropic 5 (mGluR5), nerve growth factor-␤ (NGF-␤), Jun oncogene (c-Jun), transmembrane receptor Unc5H1 (UNC5H1), and transmembrane receptor Unc5H2 (UNC5H2). All these genes, except for GH, were downregulated in response to trace conditioning. GH was upregulated; therefore, we also validated the downregulation of the GH inhibitor, somatostatin (SST), even though it just failed to meet criteria on the arrays. By during situ hybridization, GH was expressed throughout the cell layers of the hippocampus in response to trace conditioning. None of the genes regulated in trace eyeblink conditioning were similarly affected by delay conditioning, a task that does not require the hippocampus. These findings demonstrate that transcriptional profiling can exhibit a repertoire of genes sensitive to the formation of hippocampaldependent associative memories. Hippocampus 2002;12:821-833.

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Monoclonal antibodies were generated against the adenosine A1 receptor (A1R) purified from rat brain. In immunoblot analyses of purified or partially purified A1R preparations from rat brain, these antibodies recognized a solitary band, the size of which corresponded to that expected for A1R. These antibodies recognized not only the native form of A1R but also the deglycosylated form of A1R. Immunocytochemical analysis of Chinese hamster ovarian cells that were transfected stably with rat A1R cDNA showed that their cell bodies were stained intensely by these antibodies, whereas nontransfected Chinese hamster ovarian cells were not. These antibodies detected the A1R naturally present in the DDT1 MF‐2 smooth muscle cells. One of these antibodies (the 511CA antibody) was then used to examine the immunohistochemical distribution of A1Rs in rat forebrain. On light microscopy, A1R immunoreactivity was observed in the cerebral cortex, septum, basal ganglia, hippocampal formation, and thalamus. However, in some regions of the forebrain, regional differences in staining intensity were found as follows: In the cerebral cortex, the strongest immunoreactivity was found in the large pyramidal neurons of layer V. This immunoreactivity was detected in the pyramidal cell bodies, dendrites, and axon initial segments. In the hippocampus, A1R immunoreactivity was detected mainly in the stratum pyramidale. The pyramidal cells in fields CA2–CA3 of the hippocampus were stained more intensely or more clearly than those in field CA1 or the dentate gyrus. More intense A1R immunoreactivity of the apical dendrites was detected in field CA2 compared with other hippocampal fields and the dentate gyrus. Many interneurons of the hippocampus were stained by the 511CA antibody. The subcellular distribution of A1Rs in the forebrain was examined by using a digital deconvolution system and electron microscopy. In the cerebral cortex, the view obtained by removing the background haze by deconvolution revealed that the immunofluoresence‐labeled A1Rs were distributed on the surfaces of the cell bodies and dendrites and in the cytoplasm of layer V neurons as small spots. In field CA1, immunoreactivity was detected in the areas surrounding pyramidal cells. Electron microscopy revealed the presence of A1R‐immunoreactive products in both the presynaptic terminals and the postsynaptic structures. The specific cellular distribution of A1Rs is consistent with the physiological premise that endogeneously released adenosine exerts control over the excitability of forebrain neurons at both presynaptic and postsynaptic sites through A1Rs. J. Comp. Neurol. 411:301–316, 1999. © 1999 Wiley‐Liss, Inc.

The intracellular accumulation of amyloid-β (Aβ) oligomers critically contributes to disease progression in Alzheimer’s disease (AD) and can be the potential target of AD therapy. Direct observation of molecular dynamics of Aβ oligomers in vivo is key for drug discovery research, however, it has been challenging because Aβ aggregation inhibits the fluorescence from fusion proteins. Here, we developed Aβ1-42-GFP fusion proteins that are oligomerized and visualize their dynamics inside cells even when aggregated. We examined the aggregation states of Aβ-GFP fusion proteins using several methods and confirmed that they did not assemble into fibrils, but instead formed oligomers in vitro and in live cells. By arranging the length of the liker between Aβ and GFP, we generated two fusion proteins with “a long-linker” and “a short-linker”, and revealed that the aggregation property of fusion proteins can be evaluated by measuring fluorescence intensities using rat primary culture neurons transfected with Aβ-GFP plasmids and Aβ-GFP transgenic C. elegans. We found that Aβ-GFP fusion proteins induced cell death in COS7 cells. These results suggested that novel Aβ-GFP fusion proteins could be utilized for studying the physiological functions of Aβ oligomers in living cells and animals, and for drug screening by analyzing Aβ toxicity.