Different Excitation-Inhibition Correlations Between Spontaneous and Tone-evoked Activity in Primary Auditory Cortex Neurons.

Tone-evoked synaptic excitation and inhibition are highly correlated in many neurons with V-shaped tuning curves in the primary auditory cortex of pentobarbital-anesthetized rats. In contrast, there is less correlation between spontaneous excitation and inhibition in visual cortex neurons under the same anesthetic conditions. However, it was not known whether the primary auditory cortex resembles visual cortex in having spontaneous excitation and inhibition that is less correlated than tone-evoked excitation and inhibition. Here we report whole-cell voltage-clamp measurements of spontaneous excitation and inhibition in primary auditory cortex neurons of pentobarbital-anesthetized rats. Spontaneous excitatory and inhibitory currents appeared to mainly consist of distinct events, with the inhibitory event rate typically lower than the excitatory event rate. We use the ratio of the excitatory event rate to the inhibitory event rate, and the assumption that the excitatory and inhibitory synaptic currents can each be reasonably described as a filtered Poisson process, to estimate the maximum spontaneous excitatory-inhibitory correlation for each neuron. In a subset of neurons, we also measured tone-evoked excitation and inhibition. In neurons with V-shaped tuning curves, although tone-evoked excitation and inhibition were highly correlated, the spontaneous inhibitory event rate was typically sufficiently lower than the spontaneous excitatory event rate to indicate a lower excitatory-inhibitory correlation for spontaneous activity than for tone-evoked responses.

Bidirectional modulation of hippocampal synaptic plasticity by Dopaminergic D4-receptors in the CA1 area of hippocampus.

Long-term potentiation (LTP) is the persistent increase in the strength of the synapses. However, the neural networks would become saturated if there is only synaptic strenghthening. Synaptic weakening could be facilitated by active processes like long-term depression (LTD). Molecular mechanisms that facilitate the weakening of synapses and thereby stabilize the synapses are also important in learning and memory. Here we show that blockade of dopaminergic D4 receptors (D4R) promoted the formation of late-LTP and transformed early-LTP into late-LTP. This effect was dependent on protein synthesis, activation of NMDA-receptors and CaMKII. We also show that GABAA-receptor mediated mechanisms are involved in the enhancement of late-LTP. We could show that short-term plasticity and baseline synaptic transmission were unaffected by D4R inhibition. On the other hand, antagonizing D4R prevented both early and late forms of LTD, showing that activation of D4Rs triggered a dual function. Synaptic tagging experiments on LTD showed that D4Rs act as plasticity related proteins rather than the setting of synaptic tags. D4R activation by PD 168077 induced a slow-onset depression that was protein synthesis, NMDAR and CaMKII dependent. The D4 receptors, thus exert a bidirectional modulation of CA1 pyramidal neurons by restricting synaptic strengthening and facilitating synaptic weakening.

Transgenic Mice Overexpressing the Divalent Metal Transporter 1 Exhibit Iron Accumulation and Enhanced Parkin Expression in the Brain.

Exposure to divalent metals such as iron and manganese is thought to increase the risk for Parkinson's disease (PD). Under normal circumstances, cellular iron and manganese uptake is regulated by the divalent metal transporter 1 (DMT1). Accordingly, alterations in DMT1 levels may underlie the abnormal accumulation of metal ions and thereby disease pathogenesis. Here, we have generated transgenic mice overexpressing DMT1 under the direction of a mouse prion promoter and demonstrated its robust expression in several regions of the brain. When fed with iron-supplemented diet, DMT1-expressing mice exhibit rather selective accumulation of iron in the substantia nigra, which is the principal region affected in human PD cases, but otherwise appear normal. Alongside this, the expression of Parkin is also enhanced, likely as a neuroprotective response, which may explain the lack of phenotype in these mice. When DMT1 is overexpressed against a Parkin null background, the double-mutant mice similarly resisted a disease phenotype even when fed with iron- or manganese-supplemented diet. However, these mice exhibit greater vulnerability toward 6-hydroxydopamine-induced neurotoxicity. Taken together, our results suggest that iron accumulation alone is not sufficient to cause neurodegeneration and that multiple hits are required to promote PD.

Iron mitigates DMT1-mediated manganese cytotoxicity via the ASK1-JNK signaling axis: Implications of iron supplementation for manganese toxicity.

Manganese (Mn(2+)) neurotoxicity from occupational exposure is well documented to result in a Parkinson-like syndrome. Although the understanding of Mn(2+) cytotoxicity is still incomplete, both Mn(2+) and Fe(2+) can be transported via the divalent metal transporter 1 (DMT1), suggesting that competitive uptake might disrupt Fe(2+) homeostasis. Here, we found that DMT1 overexpression significantly enhanced Mn(2+) cytoplasmic accumulation and JNK phosphorylation, leading to a reduction in cell viability. Although a robust activation of autophagy was observed alongside these changes, it did not trigger autophagic cell death, but was instead shown to be essential for the degradation of ferritin, which normally sequesters labile Fe(2+). Inhibition of ferritin degradation through the neutralization of lysosomal pH resulted in increased ferritin and enhanced cytoplasmic Fe(2+) depletion. Similarly, direct Fe(2+) chelation also resulted in aggravated Mn(2+)-mediated JNK phosphorylation, while Fe(2+) repletion protected cells, and this occurs via the ASK1-thioredoxin pathway. Taken together, our study presents the novel findings that Mn(2+) cytotoxicity involves the depletion of the cytoplasmic Fe(2+) pool, and the increase in autophagy-lysosome activity is important to maintain Fe(2+) homeostasis. Thus, Fe(2+) supplementation could have potential applications in the prevention and treatment of Mn(2+)-mediated toxicity.

Proteasome inhibition promotes Parkin-Ubc13 interaction and lysine 63-linked ubiquitination.

Disruption of the ubiquitin-proteasome system, which normally identifies and degrades unwanted intracellular proteins, is thought to underlie neurodegeneration. Supporting this, mutations of Parkin, a ubiquitin ligase, are associated with autosomal recessive parkinsonism. Remarkably, Parkin can protect neurons against a wide spectrum of stress, including those that promote proteasome dysfunction. Although the mechanism underlying the preservation of proteasome function by Parkin is hitherto unclear, we have previously proposed that Parkin-mediated K63-linked ubiquitination (which is usually uncoupled from the proteasome) may serve to mitigate proteasomal stress by diverting the substrate load away from the machinery. By means of linkage-specific antibodies, we demonstrated here that proteasome inhibition indeed promotes K63-linked ubiquitination of proteins especially in Parkin-expressing cells. Importantly, we further demonstrated that the recruitment of Ubc13 (an E2 that mediates K63-linked polyubiquitin chain formation exclusively) by Parkin is selectively enhanced under conditions of proteasomal stress, thus identifying a mechanism by which Parkin could promote K63-linked ubiquitin modification in cells undergoing proteolytic stress. This mode of ubiquitination appears to facilitate the subsequent clearance of Parkin substrates via autophagy. Consistent with the proposed protective role of K63-linked ubiquitination in times of proteolytic stress, we found that Ubc13-deficient cells are significantly more susceptible to cell death induced by proteasome inhibitors compared to their wild type counterparts. Taken together, our study suggests a role for Parkin-mediated K63 ubiquitination in maintaining cellular protein homeostasis, especially during periods when the proteasome is burdened or impaired.

Enhanced autophagy from chronic toxicity of iron and mutant A53T α-synuclein: implications for neuronal cell death in Parkinson disease.

Parkinson disease (PD), a prevalent neurodegenerative motor disorder, is characterized by the rather selective loss of dopaminergic neurons and the presence of α-synuclein-enriched Lewy body inclusions in the substantia nigra of the midbrain. Although the etiology of PD remains incompletely understood, emerging evidence suggests that dysregulated iron homeostasis may be involved. Notably, nigral dopaminergic neurons are enriched in iron, the uptake of which is facilitated by the divalent metal ion transporter DMT1. To clarify the role of iron in PD, we generated SH-SY5Y cells stably expressing DMT1 either singly or in combination with wild type or mutant α-synuclein. We found that DMT1 overexpression dramatically enhances Fe(2+) uptake, which concomitantly promotes cell death. This Fe(2+)-mediated toxicity is aggravated by the presence of mutant α-synuclein expression, resulting in increased oxidative stress and DNA damage. Curiously, Fe(2+)-mediated cell death does not appear to involve apoptosis. Instead, the phenomenon seems to occur as a result of excessive autophagic activity. Accordingly, pharmacological inhibition of autophagy reverses cell death mediated by Fe(2+) overloading. Taken together, our results suggest a role for iron in PD pathogenesis and provide a mechanism underlying Fe(2+)-mediated cell death.

Parkin mediates apparent E2-independent monoubiquitination in vitro and contains an intrinsic activity that catalyzes polyubiquitination.

BACKGROUND: Mutations in the parkin gene, which encodes a ubiquitin ligase (E3), are a major cause of autosomal recessive parkinsonism. Although parkin-mediated ubiquitination was initially linked to protein degradation, accumulating evidence suggests that the enzyme is capable of catalyzing multiple forms of ubiquitin modifications including monoubiquitination, K48- and K63-linked polyubiquitination. In this study, we sought to understand how a single enzyme could exhibit such multifunctional catalytic properties. METHODS AND FINDINGS: By means of in vitro ubiquitination assays coupled with mass spectrometry analysis, we were surprised to find that parkin is apparently capable of mediating E2-independent protein ubiquitination in vitro, an unprecedented activity exhibited by an E3 member. Interestingly, whereas full length parkin catalyzes solely monoubiquitination regardless of the presence or absence of E2, a truncated parkin mutant containing only the catalytic moiety supports both E2-independent and E2-dependent assembly of ubiquitin chains. CONCLUSIONS: Our results here suggest a complex regulation of parkin's activity and may help to explain how a single enzyme like parkin could mediate diverse forms of ubiquitination.

Stress-induced alterations in parkin solubility promote parkin aggregation and compromise parkin's protective function.

Mutations in parkin are currently recognized as the most common cause of familial Parkinsonism. Emerging evidence also suggests that parkin expression variability may confer a risk for the development of the more common, sporadic form of Parkinson's disease (PD). Supporting this, we have recently demonstrated that parkin solubility in the human brain becomes altered with age. As parkin apparently functions as a broad-spectrum neuroprotectant, the resulting decrease in the availability of soluble parkin with age may underlie the progressive susceptibility of the brain to stress. Interestingly, we also observed that many familial-PD mutations of parkin alter its solubility in a manner that is highly reminiscent of our observations with the aged brain. The converging effects on parkin brought about by aging and PD-causing mutations are probably not trivial and suggest that environmental modulators affecting parkin solubility would increase an individual's risk of developing PD. Using both cell culture and in vivo models, we demonstrate here that several PD-linked stressors, including neurotoxins (MPP+, rotenone, 6-hydroxydopamine), paraquat, NO, dopamine and iron, induce alterations in parkin solubility and result in its intracellular aggregation. Furthermore, the depletion of soluble, functional forms of parkin is associated with reduced proteasomal activities and increased cell death. Our results suggest that exogenously introduced stress as well as endogenous dopamine could affect the native structure of parkin, promote its misfolding, and concomitantly compromise its protective functions. Mechanistically, our results provide a link between the influence of environmental and intrinsic factors and genetic susceptibilities in PD pathogenesis.

The 350-fold compacted Fugu parkin gene is structurally and functionally similar to human Parkin.

Mutations in the human parkin gene (huParkin) are the predominant genetic cause of familial parkinsonism. The huParkin locus, spanning about 1.4 Mb, is one of the largest in the human genome. Despite its huge size, huParkin codes for a rather short transcript of about 4.5 kb. To gain an insight into the structure, function and evolutionary history of huParkin, we have characterized the pufferfish [Fugu rubripes (Fugu)] ortholog of huParkin. A remarkable feature of the Fugu parkin gene (fuparkin) is its unusually compact size. It spans only about 4 kb and is thus 350-fold smaller than its human ortholog. The Fugu and human parkin genes are otherwise highly similar in their genomic organization and expression pattern. Furthermore, like human Parkin, Fugu parkin also functions as an ubiquitin ligase. These shared features between fuparkin and huParkin suggest that the physiological function and regulation of the parkin gene are conserved during the evolution of vertebrates. Conceivably, the compact locus of fuparkin could serve as a useful model to understand the transcriptional regulation of huParkin.

Parkin mediates nonclassical, proteasomal-independent ubiquitination of synphilin-1: implications for Lewy body formation.

It is widely accepted that the familial Parkinson's disease (PD)-linked gene product, parkin, functions as a ubiquitin ligase involved in protein turnover via the ubiquitin-proteasome system. Substrates ubiquitinated by parkin are hence thought to be destined for proteasomal degradation. Because we demonstrated previously that parkin interacts with and ubiquitinates synphilin-1, we initially expected synphilin-1 degradation to be enhanced in the presence of parkin. Contrary to our expectation, we found that synphilin-1 is normally ubiquitinated by parkin in a nonclassical, proteasomal-independent manner that involves lysine 63 (K63)-linked polyubiquitin chain formation. Parkin-mediated degradation of synphilin-1 occurs appreciably only at an unusually high parkin to synphilin-1 expression ratio or when primed for lysine 48 (K48)-linked ubiquitination. In addition we found that parkin-mediated ubiquitination of proteins within Lewy-body-like inclusions formed by the coexpression of synphilin-1, alpha-synuclein, and parkin occurs predominantly via K63 linkages and that the formation of these inclusions is enhanced by K63-linked ubiquitination. Our results suggest that parkin is a dual-function ubiquitin ligase and that K63-linked ubiquitination of synphilin-1 by parkin may be involved in the formation of Lewy body inclusions associated with PD.