CLIC1 functional expression is required for cAMP‐induced neurite elongation in post‐natal mouse retinal ganglion cells

During neuronal differentiation, axonal elongation is regulated by both external and intrinsic stimuli, including neurotropic factors, cytoskeleton dynamics, second messengers such as cyclic adenosine monophosphate (cAMP), and neuronal excitability. Chloride intracellular channel 1 (CLIC1) is a cytoplasmic hydrophilic protein that, upon stimulation, dimerizes and translocates to the plasma membrane, where it contributes to increase the membrane chloride conductance. Here, we investigated the expression of CLIC1 in primary hippocampal neurons and retinal ganglion cells (RGCs) and examined how the functional expression of CLIC1 specifically modulates neurite outgrowth of neonatal murine RGCs. Using a combination of electrophysiology and immunohistochemistry, we found that CLIC1 is expressed in hippocampal neurons and RGCs and that the chloride current mediated by CLIC1 is required for maintaining growth cone morphology and sustaining cAMP‐stimulated neurite elongation in dissociated immunopurified RGCs. In cultured RGCs, inhibition of CLIC1 ionic current through the pharmacological blocker IAA94 or a specific anti‐CLIC1 antibody directed against its extracellular domain prevents the neurite outgrowth induced by cAMP. CLIC1‐mediated chloride current, which results from an increased open probability of the channel, is detected only when cAMP is elevated. Inhibition of protein kinase A prevents such current. These results indicate that CLIC1 functional expression is regulated by cAMP via protein kinase A and is required for neurite outgrowth modulation during neuronal differentiation.

     

A HuD‐ZBP1 ribonucleoprotein complex localizes GAP‐43 mRNA into axons through its 3′ untranslated region AU‐rich regulatory element

Localized translation of axonal mRNAs contributes to developmental and regenerative axon growth. Although untranslated regions (UTRs) of many different axonal mRNAs appear to drive their localization, there has been no consensus RNA structure responsible for this localization. We recently showed that limited expression of ZBP1 protein restricts axonal localization of both β‐actin and GAP‐43 mRNAs. β‐actin 3′UTR has a defined element for interaction with ZBP1, but GAP‐43 mRNA shows no homology to this RNA sequence. Here, we show that an AU‐rich regulatory element (ARE) in GAP‐43′s 3′UTR is necessary and sufficient for its axonal localization. Axonal GAP‐43 mRNA levels increase after in vivo injury, and GAP‐43 mRNA shows an increased half‐life in regenerating axons. GAP‐43 mRNA interacts with both HuD and ZBP1, and HuD and ZBP1 co‐immunoprecipitate in an RNA‐dependent fashion. Reporter mRNA with the GAP‐43 ARE competes with endogenous β‐actin mRNA for axonal localization and decreases axon length and branching similar to the β‐actin 3′UTR competing with endogenous GAP‐43 mRNA. Conversely, over‐expressing GAP‐43 coding sequence with its 3′UTR ARE increases axonal elongation and this effect is lost when just the ARE is deleted from GAP‐43′s 3′UTR.

     

Analysis of dopaminergic neuronal dysfunction in genetic and toxin‐induced models of Parkinson's disease in Drosophila

Drosophila melanogaster has contributed significantly to the understanding of disease mechanisms in Parkinson's disease (PD) as it is one of the very few PD model organisms that allow the study of age‐dependent behavioral defects, physiology and histology, and genetic interactions among different PD‐related genes. However, there have been contradictory results from a number of recent reports regarding the loss of dopaminergic neurons in different PD fly models. In an attempt to re‐evaluate and clarify this issue, we have examined three different genetic (α‐synuclein, Pink1, parkin) and two toxin‐based (rotenone and paraquat) models of the disease for neuronal cell loss. Our results showed no dopaminergic neuronal loss in all models tested. Despite this surprising result, we found additional phenotypes showing the dysfunctional status of the dopaminergic neurons in most of the models analyzed. A common feature found in most models is a quantifiable decrease in the fluorescence of a green‐fluorescent protein reporter gene in dopaminergic neurons that correlates well with other phenotypes found for these models and can be reliably used as a hallmark of the neurodegenerative process when modeling diseases affecting the dopaminergic system in Drosophila.

     

CDK5 protects from caspase‐induced Ataxin‐3 cleavage and neurodegeneration

Spinocerebellar ataxia type 3 (SCA3) is one of at least nine inherited neurodegenerative diseases caused by an expansion of a polyglutamine tract within corresponding disease‐specific proteins. In case of SCA3, mutation of Ataxin‐3 results in aggregation of misfolded protein, formation of intranuclear as well as cytosolic inclusion bodies and cell death in distinct neuronal populations. Since cyclin‐dependent kinase‐5 (CDK5) has been shown to exert beneficial effects on aggregate formation and cell death in various polyglutamine diseases, we tested its therapeutic potential for SCA3. Our data show increased caspase‐dependent Ataxin‐3 cleavage, aggregation, and neurodegeneration in the absence of sufficient CDK5 activity. This disease‐propagating effect could be reversed by mutation of the caspase cleavage site in Ataxin‐3. Moreover, reduction of CDK5 expression levels by RNAi in vivo enhances SCA3 toxicity as assayed in a Drosophila model for SCA3. In summary, we present CDK5 as a potent neuroprotectant, regulating cleavage and thereby toxicity of Ataxin‐3 and other polyglutamine proteins.

     

Insulin and IGF1 modulate turnover of polysialylated neural cell adhesion molecule (PSA–NCAM) in a process involving specific extracellular matrix components

Cellular interactions mediated by the neural cell adhesion molecule (NCAM) are critical in cell migration, differentiation and plasticity. Switching of the NCAM‐interaction mode, from adhesion to signalling, is determined by NCAM carrying a particular post‐translational modification, polysialic acid (PSA). Regulation of cell‐surface PSA‐NCAM is traditionally viewed as a direct consequence of polysialyltransferase activity. Taking advantage of the polysialyltransferase Ca²⁺‐dependent activity, we demonstrate in TE671 cells that downregulation of PSA‐NCAM synthesis constitutes a necessary but not sufficient condition to reduce cell‐surface PSA‐NCAM; instead, PSA‐NCAM turnover required internalization of the molecule into the cytosol. PSA‐NCAM internalization was specifically triggered by collagen in the extracellular matrix (ECM) and prevented by insulin‐like growth factor (IGF1) and insulin. Our results pose a novel role for IGF1 and insulin in controlling cell migration through modulation of PSA‐NCAM turnover at the cell surface.

     

Lipocalin‐2 enhances angiogenesis in rat brain endothelial cells via reactive oxygen species and iron‐dependent mechanisms

Inflammation is a key part of central nervous system pathophysiology. However, inflammatory factors are now thought to have both beneficial and deleterious effects. Here, we examine the hypothesis that lipocalin‐2 (LCN2), an inflammatory molecule that can be up‐regulated in the distressed central nervous system, may enhance angiogenesis in brain endothelial cells. Adding LCN2 (0.5–2.0 μg/mL) to RBE (Rat brain endothelial cells). 4 rat brain endothelial cells significantly increased matrigel tube formation and scratch migration, and also elevated levels of iron and reactive oxygen species. Co‐treatment with a radical scavenger (U83836E), a Nox inhibitor (apocynin) and an iron chelating agent (deferiprone) significantly dampened the ability of LCN2 to enhance tube formation and scratch migration in brain endothelial cells. These findings provide in vitro proof of the concept that LCN2 can promote angiogenesis via iron‐ and reactive oxygen species‐related pathways, and support the idea that LCN2 may contribute to the neurovascular recovery aspects of inflammation.

     

The high‐affinity D2/D3 agonist D512 protects PC12 cells from 6‐OHDA‐induced apoptotic cell death and rescues dopaminergic neurons in the MPTP mouse model of Parkinson's disease

In this study, in vitro and in vivo experiments were carried out with the high‐affinity multifunctional D2/D3 agonist D‐512 to explore its potential neuroprotective effects in models of Parkinson's disease and the potential mechanism(s) underlying such properties. Pre‐treatment with D‐512 in vitro was found to rescue rat adrenal Pheochromocytoma PC12 cells from toxicity induced by 6‐hydroxydopamine administration in a dose‐dependent manner. Neuroprotection was found to coincide with reductions in intracellular reactive oxygen species, lipid peroxidation, and DNA damage. In vivo, pre‐treatment with 0.5 mg/kg D‐512 was protective against neurodegenerative phenotypes associated with systemic administration of MPTP, including losses in striatal dopamine, reductions in numbers of DAergic neurons in the substantia nigra (SN), and locomotor dysfunction. These observations strongly suggest that the multifunctional drug D‐512 may constitute a novel viable therapy for Parkinson's disease.

     

Hyperthermia‐induced seizures alter adenosine A1 and A2A receptors and 5′‐nucleotidase activity in rat cerebral cortex

Febrile seizure is one of the most common convulsive disorders in children. The neuromodulator adenosine exerts anticonvulsant actions through binding adenosine receptors. Here, the impact of hyperthermia‐induced seizures on adenosine A₁ and A_2A receptors and 5′‐nucleotidase activity has been studied at different periods in the cerebral cortical area by using radioligand binding, real‐time PCR, and 5′‐nucleotidase activity assays. Hyperthermic seizures were induced in 13‐day‐old rats using a warmed air stream from a hair dryer. Neonates exhibited rearing and falling over associated with hindlimb clonus seizures (stage 5 on Racine scale criteria) after hyperthermic induction. A significant increase in A₁ receptor density was observed using [³H]DPCPX as radioligand, and mRNA coding A₁ was observed 48 h after hyperthermia‐induced seizures. In contrast, a significant decrease in A_2A receptor density was detected, using [³H]ZM241385 as radioligand, 48 h after hyperthermia‐evoked convulsions. These short‐term changes in A₁ and A_2A receptors were also accompanied by a loss of 5′‐nucleotidase activity. No significant variations either in A₁ or A_2A receptor density or 5′‐nucleotidase were observed 5 and 20 days after hyperthermic seizures. Taken together, both regulation of A₁ and A_2A receptors and loss of 5′‐nucleotidase in the cerebral cortex suggest the existence of a neuroprotective mechanism against seizures.

     

Over‐expression of an inactive mutant cathepsin D increases endogenous alpha‐synuclein and cathepsin B activity in SH‐SY5Y cells

Parkinson's disease is a neurodegenerative movement disorder. The histopathology of Parkinson's disease comprises proteinaceous inclusions known as Lewy bodies, which contains aggregated α‐synuclein. Cathepsin D (CD) is a lysosomal protease previously demonstrated to cleave α‐synuclein and decrease its toxicity in both cell lines and mouse brains in vivo. Here, we show that pharmacological inhibition of CD, or introduction of catalytically inactive mutant CD, resulted in decreased CD activity and increased cathepsin B activity, suggesting a possible compensatory response to inhibition of CD activity. However, this increased cathepsin B activity was not sufficient to maintain α‐synuclein degradation, as evidenced by the accumulation of endogenous α‐synuclein. Interestingly, the levels of LC3, LAMP1, and LAMP2, proteins involved in autophagy‐lysosomal activities, as well as total lysosomal mass as assessed by LysoTracker flow cytometry, were unchanged. Neither autophagic flux nor proteasomal activities differs between cells over‐expressing wild‐type versus mutant CD. These observations point to a critical regulatory role for that endogenous CD activity in dopaminergic cells in α‐synuclein homeostasis which cannot be compensated for by increased Cathepsin B. These data support the potential need to enhance CD function in order to attenuate α‐synuclein accumulation as a therapeutic strategy against development of synucleinopathy.

     

Changes in mitochondrial morphology induced by calcium or rotenone in primary astrocytes occur predominantly through ros‐mediated remodeling

Morphological changes in mitochondria have been primarily attributed to fission and fusion, while the more pliable transformations of mitochondria (remodeling, rounding, or stretching) have been largely overlooked. In this study, we quantify the contributions of fission and remodeling to changes in mitochondrial morphology induced by the Ca²⁺ ionophore 4Br‐A23187 and the metabolic toxin rotenone. We also examine the role of reactive oxygen species (ROS) in the regulation of mitochondrial remodeling. In agreement with our previous studies, mitochondrial remodeling, not fission, is the primary contributor to Ca²⁺‐mediated changes in mitochondrial morphology induced by 4Br‐A23187 in rat cortical astrocytes. Treatment with rotenone produced similar results. In both paradigms, remodeling was selectively blocked by antioxidants whereas fission was not, suggesting a ROS‐mediated mechanism for mitochondrial remodeling. In support of this hypothesis, inhibition of endogenous ROS by overnight incubation in antioxidants resulted in elongated reticular networks of mitochondria. Examination of inner and outer mitochondrial membranes revealed that they largely acted in concert during the remodeling process . While mitochondrial morphology is traditionally ascribed to a net output of fission and fusion processes, in this study we provide evidence that the acute pliability of mitochondria can be a dominant factor in determining their morphology. More importantly, our results suggest that the remodeling process is independently regulated through a ROS‐signaling mechanism.

     

Alpha‐ketoglutarate dehydrogenase complex‐dependent succinylation of proteins in neurons and neuronal cell lines

Reversible post‐translation modifications of proteins are common in all cells and appear to regulate many processes. Nevertheless, the enzyme(s) responsible for the alterations and the significance of the modification are largely unknown. Succinylation of proteins occurs and causes large changes in the structure of proteins; however, the source of the succinyl groups, the targets, and the consequences of these modifications on other proteins remain unknown. These studies focused on succinylation of mitochondrial proteins. The results demonstrate that the α‐ketoglutarate dehydrogenase complex (KGDHC) can serve as a trans‐succinylase that mediates succinylation in an α‐ketoglutarate‐dependent manner. Inhibition of KGDHC reduced succinylation of both cytosolic and mitochondrial proteins in cultured neurons and in a neuronal cell line. Purified KGDHC can succinylate multiple proteins including other enzymes of the tricarboxylic acid cycle leading to modification of their activity. Inhibition of KGDHC also modifies acetylation by modifying the pyruvate dehydrogenase complex. The much greater effectiveness of KGDHC than succinyl‐CoA suggests that the catalysis owing to the E2k succinyltransferase is important. Succinylation appears to be a major signaling system and it can be mediated by KGDHC.

     

Regulation of RAGE splicing by hnRNP A1 and Tra2β‐1 and its potential role in AD pathogenesis

The receptor for advanced glycation end products (RAGE) gene expresses two major alternative splicing isoforms, full‐length membrane‐bound RAGE (mRAGE) and secretory RAGE (esRAGE). Both isoforms play important roles in Alzheimer's disease (AD) pathogenesis, either via interaction of mRAGE with β‐amyloid peptide (Aβ) or inhibition of the mRAGE‐activated signaling pathway. In the present study, we showed that heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and Transformer2β‐1 (Tra2β‐1) were involved in the alternative splicing of mRAGE and esRAGE. Functionally, two factors had an antagonistic effect on the regulation. Glucose deprivation induced an increased ratio of mRAGE/esRAGE via up‐regulation of hnRNP A1 and down‐regulation of Tra2β‐1. Moreover, the ratios of mRAGE/esRAGE and hnRNP A1/Tra2β‐1 were increased in peripheral blood mononuclear cells from AD patients. The results provide a molecular basis for altered splicing of mRAGE and esRAGE in AD pathogenesis.

     

The effect of Cyclin‐dependent kinase 5 on voltage‐dependent calcium channels in PC12 cells varies according to channel type and cell differentiation state

Cyclin‐dependent kinase 5 (Cdk5) is a Ser/Thr kinase that plays an important role in the release of neurotransmitter from pre‐synaptic terminals triggered by Ca²⁺ influx into the pre‐synaptic cytoplasm through voltage‐dependent Ca²⁺ channels (VDCCs). It is reported that Cdk5 regulates L‐, P/Q‐, or N‐type VDCC, but there is conflicting data as to the effect of Cdk5 on VDCC activity. To clarify the mechanisms involved, we examined the role of Cdk5 in regulating the Ca²⁺‐channel property of VDCCs, using PC12 cells expressing endogenous, functional L‐, P/Q‐, and N‐type VDCCs. The Ca²⁺ influx, induced by membrane depolarization with high K⁺, was monitored with a fluorescent Ca²⁺ indicator protein in both undifferentiated and nerve growth factor (NGF)‐differentiated PC12 cells. Overall, Ca²⁺ influx was increased by expression of Cdk5‐p35 in undifferentiated PC12 cells but suppressed in differentiated PC12 cells. Moreover, we found that different VDCCs are distinctly regulated by Cdk5‐p35 depending on the differentiation states of PC12 cells. These results indicate that Cdk5‐p35 regulates L‐, P/Q‐, or N‐type VDCCs in a cellular context‐dependent manner.