Aggregates In Neuronal Cells Research Articles

Pirh2 modulates amyloid-β aggregation through the regulation of glucose-regulated protein 78 and chaperone-mediated signaling.

Amyloid-β (Aβ) protein aggregation in the brain is a pathological hallmark of Alzheimer's disease (AD) however, the underlying molecular mechanisms regulating amyloid aggregation are not well understood. Here, we studied the propitious role of E3 ubiquitin ligase Pirh2 in Aβ protein aggregation in view of its regulatory ligase activity in the ubiquitin-proteasome systememploying both cellular and sporadic rodent models of AD. Pirh2 protein abundance was significantly increased during Streptozotocin (STZ) induced AD conditions, and transient silencing of Pirh2 significantly inhibited the Aβ aggregation and modified the dendrite morphology along with the substantial decrease in choline level in the differentiated neurons. MALDI-TOF/TOF, coimmunoprecipitation, and UbcH7-linked in vitro ubiquitylation analysis confirmed the high interaction of Pirh2 with chaperone GRP78. Furthermore, Pirh2 silencing inhibits the STZ induced altered level of endoplasmic reticulum stress and intracellular Ca2+ levels in neuronal N2a cells. Pirh2 silencing also inhibited the AD conditions related to the altered protein abundance of HSP90 and its co-chaperones which may collectively involve in the reduced burden of amyloid aggregates in neuronal cells. Pirh2 silencing further stabilized the nuclear translocation of phospho-Nrf2 and inhibited the altered level of autophagy factors. Taken together, our data indicated that Pirh2 is critically involved in STZ induced AD pathogenesis through its interaction with ER-chaperone GRP78, improves the neuronal connectivity, affects the altered level of chaperones, co-chaperones, & autophagic markers, and collectively inhibits the Aβ aggregation.

Insights into the Structural Conformations of the Tau Protein in Different Aggregation Status.

Tau is a protein characterized by large structural portions displaying extended conformational changes. Unfortunately, the accumulation of this protein into toxic aggregates in neuronal cells leads to a number of severe pathologies, collectively named tauopathies. In the last decade, significant research advancements were achieved, including a better understanding of Tau structures and their implication in different tauopathies. Interestingly, Tau is characterized by a high structural variability depending on the type of disease, the crystallization conditions, and the formation of pathologic aggregates obtained from in vitro versus ex vivo samples. In this review, we reported an up-to-date and comprehensive overview of Tau structures reported in the Protein Data Bank, with a special focus on discussing the connections between structural features, different tauopathies, different crystallization conditions, and the use of in vitro or ex vivo samples. The information reported in this article highlights very interesting links between all these aspects, which we believe may be of particular relevance for a more informed structure-based design of compounds able to modulate Tau aggregation.

Dephosphorylation Targeting Chimaera (DEPTAC): Targeting Tau Proteins in Tauopathies.

One salient hallmark of neurodegeneration is the accumulation of toxic protein aggregates in neuronal cells. This proteotoxicity culminates in the deterioration of neuronal function. In AD and related tauopathies, the microtubule-associated protein tau becomes hyperphosphorylated. Hyperphosphorylated tau forms neurofibrillary tangles (NFTs) within neurons, which constitute a unique feature of tauopathies, including AD. A recent study has exploited a novel molecular strategy to counteract hyperphosphorylated tau and enhance its degradation. Analogous to the PROTAC methodology, a novel dephosphorylation targeting chimera (DEPTAC) was designed to promote the molecular interaction between tau and phosphatase, which, in turn, augments its degradation. Herein, we briefly discuss this novel finding and its potential therapeutic implications.

Lysosomal peptidases-intriguing roles in cancer progression and neurodegeneration.

Lysosomal peptidases are hydrolytic enzymes capable of digesting waste proteins that are targeted to lysosomes via endocytosis and autophagy. Besides intracellular protein catabolism, they play more specific roles in several other cellular processes and pathologies, either within lysosomes, upon secretion into the cell cytoplasm or extracellular space, or bound to the plasma membrane. In cancer, lysosomal peptidases are generally associated with disease progression, as they participate in crucial processes leading to changes in cell morphology, signaling, migration, and invasion, and finally metastasis. However, they can also enhance the mechanisms resulting in cancer regression, such as apoptosis of tumor cells or antitumor immune responses. Lysosomal peptidases have also been identified as hallmarks of aging and neurodegeneration, playing roles in oxidative stress, mitochondrial dysfunction, abnormal intercellular communication, dysregulated trafficking, and the deposition of protein aggregates in neuronal cells. Furthermore, deficiencies in lysosomal peptidases may result in other pathological states, such as lysosomal storage disease. The aim of this review was to highlight the role of lysosomal peptidases in particular pathological processes of cancer and neurodegeneration and to address the potential of lysosomal peptidases in diagnosing and treating patients.

Effects of doxorubicin on the developing brain of albino mice embryos

Introduction: Doxorubicin is an anthracycline antineoplastic drug. It has been widely used in the treatment of many malignancies including haematological malignancies, soft tissues sarcomas, breast cancer and neuro-blastoma etc. The doxorubicin rat model is an established model which encompasses spectrum of anomalies seen in VACTERAL associations and has similarities to human VACTER anomalies. The aim of present study is to support the anomalies produced by doxorubicin in albino mice embryos. Material and Methods: The present study was conducted in the department of anatomy, IMS, BHU Varanasi, U.P, on fifty female albino mice of an average age of 80 to 100 days, kept in plastic cage with male (3female:1male) in aseptic air condition. After confirming the pregnancy, the drug was administered intraperitoneally on the 8th day of gestation in a dose of 3mg/kg body weight. The foetuses were collected by exteriorization of uterine horn on 19th day of gestation and examined for congenital anomalies. Observations:Foetuses were divided into two groups, control and treated. Out of 239 treated foetuses, 25 foetuses were found dead, 106 foetuses showed congenital anomalies and the rest 108 foetuses were found normal. The brains of treated foetuses were smaller and reduced in weight (microcephaly) as compared to average weight and size of brain of control foetuses. On histological examination, cerebrum and cerebellum showed degeneration and aggregation of neuronal cells. Conclusions:The drug was found to be lethal as well as teratogenic in developing mice embryos. adriamycine rat model for VACTERL anomalies. Since, mice are the foremost mammal model for researchers; the present study also establishes doxorubicin mouse model support to the well established. Keywords:Doxorubicin (Adriamycine), VACTERL (Vertebral, anal, Cardiac, Tracheal, Oesophageal, Renal & limb) anomalies.

Fate and propagation of endogenously formed Tau aggregates in neuronal cells

Tau accumulation in the form of neurofibrillary tangles in the brain is a hallmark of tauopathies such as Alzheimer's disease (AD). Tau aggregates accumulate in brain regions in a defined spatiotemporal pattern and may induce the aggregation of native Tau in a prion‐like manner. However, the underlying mechanisms of cell‐to‐cell spreading of Tau pathology are unknown and could involve encapsulation within exosomes, trans‐synaptic passage, and tunneling nanotubes (TNTs). We have established a neuronal cell model to monitor both internalization of externally added fibrils, synthetic (K18) or Tau from AD brain extracts, and real‐time conversion of microtubule‐binding domain of Tau fused to a fluorescent marker into aggregates. We found that these endogenously formed deposits colabel with ubiquitin and p62 but are not recruited to macroautophagosomes, eventually escaping clearance. Furthermore, endogenous K18‐seeded Tau aggregates spread to neighboring cells where they seed new deposits. Transfer of Tau aggregates depends on direct cell contact, and they are found inside TNTs connecting neuronal cells. We further demonstrate that contact‐dependent transfer occurs in primary neurons and between neurons and astrocytes in organotypic cultures.

SCF-Slimb is critical for Glycogen synthase kinase-3β-mediated suppression of TAF15-induced neurotoxicity in Drosophila.

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disorder characterized pathologically by motor neuron degeneration and associated with aggregation of RNA-binding proteins. TATA-binding protein-associated factor 15 (TAF15) accumulates as cytoplasmic aggregates in neuronal cells, and clearance of these aggregates is considered a potential therapeutic strategy for ALS. However, the exact pathogenic mechanism of TAF15-induced neurotoxicity remains to be elucidated. Glycogen synthase kinase-3 (GSK-3) plays a critical role in the protection of ALS pathology. In the present study, we use a transgenic fly model over-expressing human TAF15 to study the protective effects of Shaggy/GSK3β on TAF15-induced neuronal toxicity in Drosophila brain. Transgenic flies were examined for locomotor activity and lithium treatment. The expression level and solubility of TAF15 were assessed with western blotting, whereas immunohistochemistry was used to assess TAF15 aggregation in Drosophila brain. We have revealed that Shaggy/GSK3β was abnormally activated in neurons of TAF15-expressing flies and its inhibition can suppress the defective phenotypes, thereby preventing retinal degeneration and locomotive activity caused by TAF15. We have also found that Shaggy/GSK3β inhibition in neuronal cells leads to a reduction in TAF15 levels. Indeed, the F-box proteins Slimb and archipelago genetically interact with TAF15 and control TAF15 protein level in Drosophila. Importantly, SCFslimb is a critical regulator for Shaggy/GSK3β-mediated suppression of TAF15-induced toxicity in Drosophila. The present study has provided an in vivo evidence supporting the molecular mechanism of GSK3β inhibition for protection against TAF15-linked proteinopathies.

Exercise‐enriched extracellular vesicles benefit brain health by decreasing protein aggregation

Aerobic exercise results in improved brain health during both healthy aging and dementia. However, the precise biological mechanisms that drive exercise‐induced benefits for brain health remain unknown. One possible mechanism by which exercise may benefit the brain is by facilitating the delivery of molecular mediators within extracellular vesicles (EVs; exosomes and microvesicles). EVs are bilayer‐phospholipid enclosed vesicles that carry protein and microRNA cargo and are released into the blood from many organs and cells. EVs have been shown to regulate nerve regeneration, synaptic function and behavior. Importantly, when directly injected into rodent brains, EVs can effectively eliminate protein aggregates like amyloid‐β‐peptide (Aβ), which are closely linked to Alzheimer’s Disease (AD) pathology. We hypothesize that EVs isolated from human serum post‐exercise could effectively clear Aβ aggregates in neuronal cells. To test this hypothesis, we isolated EVs from cognitively healthy subjects and early onset AD subjects (8 non‐dementia subjects, ND and 4 early AD subjects) 15 minutes after maximal aerobic exercise (EX). EV size did not change with exercise in either ND or AD subjects, while EV concentration increased by 20% post‐EX in both groups. EV function was tested by incubating serum EVs with human neuronal SY5Y cells over expressing β‐amyloid precursor protein (APPswe). We found that post‐EX‐derived EVs were more effective at alleviating Aβ protein aggregation (40% decrease in aggregation compared to pre‐EX‐derived EVs). Heat Shock Proteins (HSPs), molecular chaperones that play a critical role in protein folding/misfolding, were increased in post‐EX EVs. These findings demonstrate a novel way in which exercise could benefit the brain, through the cell‐to‐cell signaling network of extracellular vesicle communication.Support or Funding InformationResearch support by the University of Kansas Alzheimer’s Disease Center.

Chaperone Mediated Autophagy Degrades TDP-43 Protein and Is Affected by TDP-43 Aggregation.

TAR DNA binding protein 43 kDa (TDP-43) is a ribonuclear protein regulating many aspects of RNA metabolism. Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD) are fatal neurodegenerative diseases with the presence of TDP-43 aggregates in neuronal cells. Chaperone Mediated Autophagy (CMA) is a lysosomal degradation pathway participating in the proteostasis of several cytosolic proteins including neurodegenerative associated proteins. In addition, protein oligomers or aggregates can affect the status of CMA. In this work, we studied the relationship between CMA and the physiological and pathological forms of TDP-43. First, we found that recombinant TDP-43 was specifically degraded by rat liver’s CMA+ lysosomes and that endogenous TDP-43 is localized in rat brain’s CMA+ lysosomes, indicating that TDP-43 can be a CMA substrate in vivo. Next, by using a previously reported TDP-43 aggregation model, we have shown that wild-type and an aggregate-prone form of TDP-43 are detected in CMA+ lysosomes isolated from cell cultures. In addition, their protein levels increased in cells displaying CMA down-regulation, indicating that these two TDP-43 forms are CMA substrates in vitro. Finally, we observed that the aggregate-prone form of TDP-43 is able to interact with Hsc70, to co-localize with Lamp2A, and to up-regulate the levels of these molecular components of CMA. The latter was followed by an up-regulation of the CMA activity and lysosomal damage. Altogether our data shows that: (i) TDP-43 is a CMA substrate; (ii) CMA can contribute to control the turnover of physiological and pathological forms of TDP-43; and (iii) TDP-43 aggregation can affect CMA performance. Overall, this work contributes to understanding how a dysregulation between CMA and TDP-43 would participate in neuropathological mechanisms associated with TDP-43 aggregation.

Dynamic Interaction of USP14 with the Chaperone HSC70 Mediates Crosstalk between the Proteasome, ER Signaling, and Autophagy.

SummaryUSP14 is a deubiquitinating enzyme associated with the proteasome important for protein degradation. Here we show that upon proteasome inhibition or expression of the mutant W58A-USP14, association of USP14 with the 19S regulatory particle is disrupted. MS-based interactomics revealed an interaction of USP14 with the chaperone, HSC70, in neuroblastoma cells. Proteasome inhibition enhanced binding of USP14 to HSC70, and to XBP1u and IRE1α proteins, demonstrating a role in the unfolded protein response. Striatal neurons expressing mutant huntingtin exhibited reduced USP14 and HSC70 levels, whereas inhibition of HSC70 downregulated USP14. Furthermore, proteasome inhibition or use of the mutant W58A-USP14 facilitated the interaction of USP14 with the autophagy protein, GABARAP. Functionally, overexpression of W58A-USP14 increased GABARAP positive autophagosomes in striatal neurons, and this was abrogated using the HSC70 inhibitor, VER-155008. Modulation of the USP14-HSC70 axis may represent a potential therapeutic target in HD to beneficially influence multiple proteostasis pathways.

Genetic activation of parkin rescues TAF15-induced neurotoxicity in a Drosophila model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder that is characterized pathologically by the loss of motor neurons. Mutations in the TAF15 gene have been implicated in the pathogenesis of ALS. TATA-binding protein associated factor 15 (TAF15) accumulates as cytoplasmic aggregates in neuronal cells, the clearance of which may be a therapeutic strategy for ALS. However, the identification of a novel regulator for protection against a TAF15-induced proteinopathy and the exact pathogenic mechanism of TAF15-induced neurodegeneration remain to be elucidated. Here, we show that parkin directly binds to TAF15 and that parkin overexpression can suppress the defective phenotypes, including the life span and locomotive activity of a TAF15-induced proteinopathy. We also found that overexpression of parkin in neuronal cells leads to a reduction in TAF15 levels, because of the E3 ubiquitin ligase activity of parkin. Our study provides invivo evidence supporting the use of parkin for neuroprotection in a TAF15-induced proteinopathy and offers new insights into the pathogenic mechanisms underlying TAF15-induced ALS.

Hybrid Multifunctional Modulators Inhibit Multifaceted Aβ Toxicity and Prevent Mitochondrial Damage.

Amyloid beta (Aβ) aggregation is the key trait responsible for the pathological devastation caused by Alzheimer's disease (AD). Among the various pathways of multifaceted toxicity exhibited by Aβ aggregates in neuronal cells, generation of reactive oxygen species (ROS) by Aβ-CuII complex and mitochondrial damage are prominent. Aβ interferes with mitochondrial transport channels, causing mitochondrial dysfunction. Herein, we present nontoxic hybrid multifunctional modulators (HMMs, TGR86-88) developed by integrating the structural and functional features of the metal chelating aggregation modulator, clioquinol (Clq), and the antioxidant epigallocatechin gallate (EGCG). Detailed biophysical and docking studies show that TGR86 interacts with Aβ and efficiently modulates both metal-dependent and metal-independent Aβ aggregation. TGR86 complexes with CuII, arrests its redox cycle, and thereby prevents the generation of ROS. The antioxidant nature of the HMMs effectively prevents DNA damage and protein oxidation. TGR86 rescued PC12 cells from Aβ-induced neurotoxicity by preventing the generation of ROS and foiling the interaction of toxic Aβ species with mitochondria, thereby averting its damage. These key attributes make TGR86 a potential candidate to develop therapeutics for the multifactorial Aβ toxicity in Alzheimer's disease.

Pimozide reduces toxic forms of tau in TauC3 mice via 5' adenosine monophosphate-activated protein kinase-mediated autophagy.

In neurodegenerative diseases like Alzheimer's disease (AD), tau is hyperphosphorylated and forms aggregates and neurofibrillary tangles in affected neurons. Autophagy is critical to clear the aggregates of disease-associated proteins and is often altered in patients and animal models of AD. Because mechanistic target of rapamycin (mTOR) negatively regulates autophagy and is hyperactive in the brains of patients with AD, mTOR is an attractive therapeutic target for AD. However, pharmacological strategies to increase autophagy by targeting mTOR inhibition cause various side effects. Therefore, autophagy activation mediated by non-mTOR pathways is a new option for autophagy-based AD therapy. Here, we report that pimozide activates autophagy to rescue tau pathology in an AD model. Pimozide increased autophagic flux through the activation of the AMPK-Unc-51 like autophagy activating kinase 1 (ULK1) axis, but not of mTOR, in neuronal cells, and this function was independent of dopamine D2 receptor inhibition. Pimozide reduced levels of abnormally phosphorylated tau aggregates in neuronal cells. Further, daily intraperitoneal (i.p.) treatment of pimozide led to a recovery from memory deficits of TauC3 mice expressing a caspase-cleaved form of tau. In the brains of these mice, we found increased phosphorylation of AMPK1 and ULK1, and reduced levels of the soluble oligomers and NP40-insoluble aggregates of abnormally phosphorylated tau. Together, these results suggest that pimozide rescues memory impairments in TauC3 mice and reduces tau aggregates by increasing autophagic flux through the mTOR-independent AMPK-ULK1 axis.

The ubiquitin signal and autophagy: an orchestrated dance leading to mitochondrial degradation

The quality of mitochondria, essential organelles that produce ATP and regulate numerous metabolic pathways, must be strictly monitored to maintain cell homeostasis. The loss of mitochondrial quality control systems is acknowledged as a determinant for many types of neurodegenerative diseases including Parkinson's disease (PD). The two gene products mutated in the autosomal recessive forms of familial early-onset PD, Parkin and PINK1, have been identified as essential proteins in the clearance of damaged mitochondria via an autophagic pathway termed mitophagy. Recently, significant progress has been made in understanding how the mitochondrial serine/threonine kinase PINK1 and the E3 ligase Parkin work together through a novel stepwise cascade to identify and eliminate damaged mitochondria, a process that relies on the orchestrated crosstalk between ubiquitin/phosphorylation signaling and autophagy. In this review, we highlight our current understanding of the detailed molecular mechanisms governing Parkin-/PINK1-mediated mitophagy and the evidences connecting Parkin/PINK1 function and mitochondrial clearance in neurons.

Self-assembly and sequence length dependence on nanofibrils of polyglutamine peptides

Huntington's disease (HD) is recognized as a currently incurable, inherited neurodegenerative disorder caused by the accumulation of misfolded polyglutamine (polyQ) peptide aggregates in neuronal cells. Yet, the mechanism by which newly formed polyQ chains interact and assemble into toxic oligomeric structures remains a critical, unresolved issue. In order to shed further light on the matter, our group elected to investigate the folding of polyQ peptides – examining glutamine repeat lengths ranging from 3 to 44 residues. To characterize these aggregates we employed a diverse array of technologies, including: nuclear magnetic resonance; circular dichroism; Fourier transform infrared spectroscopy; fluorescence resonance energy transfer (FRET), and atomic force microscopy. The data we obtained suggest that an increase in the number of glutamine repeats above 14 residues results in disordered loop structures, with different repeat lengths demonstrating unique folding characteristics. This differential folding manifests in the formation of distinct nano-sized fibrils, and on this basis, we postulate the idea of 14 polyQ repeats representing a critical loop length for neurotoxicity – a property that we hope may prove amenable to future therapeutic intervention. Furthermore, FRET measurements on aged assemblages indicate an increase in the end-to-end distance of the peptide with time, most probably due to the intermixing of individual peptide strands within the nanofibril. Further insight into this apparent time-dependent reorganization of aggregated polyQ peptides may influence future disease modeling of polyQ-related proteinopathies, in addition to directing novel clinical innovations.

Transglutaminase 6 interacts with polyQ proteins and promotes the formation of polyQ aggregates

A common feature of polyglutamine (polyQ) diseases is the presence of aggregates in neuronal cells caused by expanded polyglutamine tracts. PolyQ proteins are the substrates of transglutaminase 2, and the increased activity of transglutaminase in polyQ diseases suggests that transglutaminase may be directly involved in the formation of the aggregates. We previously identified the transglutaminase 6 gene to be causative of spinocerebellar ataxia type 35 (SCA35), and we found that SCA35-associated mutants exhibited reduced transglutaminase activity. Here we report that transglutaminase 6 interacts and co-localizes with both normal and expanded polyQ proteins in HEK293 cells. Moreover, the overexpression of transglutaminase 6 promotes the formation of polyQ aggregates and the conversion of soluble polyQ into insoluble polyQ aggregates. However, SCA35-associated mutants do not affect their interactions with polyQ proteins. These data suggest that transglutaminase 6 could be involved in polyQ diseases and there may exist a common pathological link between polyQ associated SCA and SCA35.

Transfer of polyglutamine aggregates in neuronal cells occurs in tunneling nanotubes

Huntington's disease (HD) is a dominantly inherited neurodegenerative disease caused by CAG expansion in the huntingtin gene, which adds a homopolymeric tract of polyglutamine (polyQ) to the encoded protein leading to the formation of toxic aggregates. Despite rapidly accumulating evidences supporting a role for intercellular transmission of protein aggregates, little is known about whether and how huntingtin (Htt) misfolding progresses through the brain. It has been recently reported that synthetic polyQ peptides and recombinant fragments of mutant Htt are readily internalized in cell cultures and able to seed polymerization of a reporter wild-type Htt. However, there is no direct evidence of aggregate transfer between cells and the mechanism has not been explored. By expressing recombinant fragments of mutant Htt in neuronal cells and in primary neurons, we found that aggregated fragments formed within one cell spontaneously transfer to neighbors in cell culture. We demonstrate that the intercellular spreading of the aggregates requires cell-cell contact and does not occur upon aggregate secretion. Interestingly, we found that the expression of mutant, but not wild-type Htt fragments, increases the number of tunneling nanotubes, which in turn provide an efficient mechanism of transfer.

SIRT2 interferes with autophagy-mediated degradation of protein aggregates in neuronal cells under proteasome inhibition

Abnormal protein aggregates have been suggested as a common pathogenesis of many neurodegenerative diseases. Two well-known protein degradation pathways are responsible for protein homeostasis by balancing protein biosynthesis and degradative processes: the ubiquitin–proteasome system (UPS) and autophagy-lysosomal system. UPS serves as the primary route for degradation of short-lived proteins, but large-size protein aggregates cannot be degraded by UPS. Autophagy is a unique cellular process that facilitates degradation of bulky protein aggregates by lysosome. Recent studies have demonstrated that autophagy plays a crucial role in the pathogenesis of neurodegenerative diseases characterized by abnormal protein accumulation, suggesting that regulation of autophagy may be a valuable therapeutic strategy for the treatment of various neurodegenerative diseases. Sirtuin-2 (SIRT2) is a class III histone deacetylase that is expressed abundantly in aging brain tissue. Here, we report that SIRT2 increases protein accumulation in murine cholinergic SN56 cells and human neuroblastoma SH-SY5Y cells under proteasome inhibition. Overexpression of SIRT2 inhibits lysosome-mediated autophagic turnover by interfering with aggresome formation and also makes cells more vulnerable to accumulated protein-mediated cytotoxicity by MG132 and amyloid beta. Moreover, MG132-induced accumulation of ubiquitinated proteins and p62 as well as cytotoxicity are attenuated in siRNA-mediated SIRT2-silencing cells. Taken together, these results suggest that regulation of SIRT2 could be a good therapeutic target for a range of neurodegenerative diseases by regulating autophagic flux.

Inhibition of neuronal cell–cell adhesion measured by the microscopic aggregation assay and impedance sensing

Microscopic aggregation assay and impedance sensing (IS) were used to monitor a change in in vitro neuron–neuron adhesion in response to blocking of cell adhesion molecules. By blocking neuron–neuron adhesion, migration and aggregation of neuronal cells can be inhibited. This leads to better control of spatial arrangement of cells in culture. In the literature N-CAM, L1 and N-cadherin proteins are pointed out as main regulators of neuronal adhesion. In this study, these three main cell adhesion molecules were used to inhibit neuron-to-neuron adhesion and aggregation. Both soluble extracellular domains and antigen antibodies were added to these adhesion molecules. They were investigated for their blocking ability in neuronal cultures. First, in a 96 h aggregation assay on a low-adhesive substrate, the effect of inhibition of the three proteins on aggregation of cortical neurons was investigated optically. Both L1 antibody and L1 protein had no effect on the degree of aggregation. An N-cadherin antibody however was shown to be effective in aggregation inhibition at concentrations of 1 and 3 µg ml−1. Up to 96 h no aggregation occurred. A similar effect was achieved by the N-cadherin protein, although less distinct. N-CAM blocking revealed no inhibition of aggregation. Second, results from IS corresponded to those of the aggregation assays. In these experiments neuron–neuron adhesion was also inhibited by blocking N-CAM L1 and N-cadherin. Cortical neurons were cultured in small wells containing circular 100 µm diameter gold electrodes, so small changes in cell–cell interactions in monolayers of neurons could be monitored by IS. Impedances of neuron-covered electrodes were significantly lower in the presence of the N-cadherin antibody and protein at concentrations of 1, 3 and 10 µg ml−1, indicating a less profound binding between adjacent neurons. Results from the aggregation assays and impedance measurements demonstrate the applicability of blocking cell adhesion molecules for inhibition of cell–cell adhesion and aggregation.

Overexpression of DSCR1 blocks zinc-induced neuronal cell death through the formation of nuclear aggregates

The Down syndrome (DS) candidate region gene 1 (DSCR1) is localized near DS critical region on chromosome 21 and is overexpressed in the brains of DS patients. Although DSCR1 was known for a modulator of calcineurin, the overexpression of DSCR1 is thought to play a role in neuronal cell death. Zinc, one of the most abundant transition metals in the brain, may also contribute to selective neuronal cell death when present in excessive amounts. In the present study, we investigated the effect of DSCR1 overexpression on zinc-induced cell death in hippocampal neuroprogenitor cells. The overexpression of DSCR1 caused apoptotic cell death without an apparent formation of intracellular protein inclusions. Upon exposure to zinc, soluble DSCR1 levels were significantly decreased and insoluble levels were enhanced to a similar extent, which were partially caused by the zinc-induced inhibition of proteasomal activity and a consequently diminished degradation of DSCR1. Furthermore, zinc treatment induced the formation of nuclear DSCR1 aggregates, which blocked zinc-induced cell death. These findings indicate that, although the up-regulation of DSCR1 levels exerts a cytotoxic effect, the addition of zinc leads to the formation of cytoprotective nuclear aggregates in neuronal cells.