p35/Cyclin-dependent kinase 5 is required for protection against beta-amyloid-induced cell death but not tau phosphorylation by ceramide.

Ceramide is a bioactive sphingolipid that can prevent calpain activation and beta-amyloid (A beta) neurotoxicity in cortical neurons. Recent evidence supports A beta induction of a calpain-dependent cleavage of the cyclin-dependent kinase 5 (cdk5) regulatory protein p35 that contributes to tau hyperphosphorylation and neuronal death. Using cortical neurons isolated from wild-type and p35 knockout mice, we investigated whether ceramide required p35/cdk5 to protect against A beta-induced cell death and tau phosphorylation. Ceramide inhibited A beta-induced calpain activation and cdk5 activity in wild-type neurons and protected against neuronal death and tau hyperphosphorylation. Interestingly, A beta also increased cdk5 activity in p35-/- neurons, suggesting that the alternate cdk5 regulatory protein, p39, might mediate this effect. In p35 null neurons, ceramide blocked A beta-induced calpain activation but did not inhibit cdk5 activity or cell death. However, ceramide blocked tau hyperphosphorylation potentially via inhibition of glycogen synthase kinase-3beta. These data suggest that ceramide can regulate A beta cell toxicity in a p35/cdk5-dependent manner.

beta-Amyloid and endoplasmic reticulum stress responses in primary neurons: effects of drugs that interact with the cytoskeleton.

In vitro studies designed to probe the cellular mechanisms underlying beta-amyloid (Abeta) toxicity in neurons have implicated several processes, including hyperphosphorylation of the microtubule (MT)-associated protein tau, loss of MT stability, and increased cytosolic calcium levels. Given that Alzheimer's disease involves accumulation of aggregates of two different proteins, the potential involvement of the unfolded protein response (UPR) and endoplasmic reticulum (ER) dysfunction has been suggested to lead to cell death. The relationship between these apparently divergent factors and pathways in Abeta toxicity is still unclear. In these studies we investigated the relationship between MT stability and the ER stress response in primary neurons exposed to toxic Abeta peptides in culture. In addition, nocodazole (ND) was used to determine if direct disruption of MT organization activated the UPR. Pretreatment of neurons with MT-stabilizing drugs paclitaxel (Taxol) and epothilone A prevented the induction of three indicators of the UPR induced by Abeta, ND, and thapsigargin, a compound known to inhibit the sarco-ER Ca(2+)-ATPase and deplete ER calcium stores, resulting in initiation of the UPR. In addition, treatment with MT-stabilizing drugs blocked cell death and the cytoskeletal disorganization induced by these insults. The results suggest that loss of cytoskeletal integrity is a very early step in the response to a variety of toxic stimuli and that preservation of MT stability might be important in preventing the induction of ER dysfunction and subsequent cell death by Abeta in neurons.

Small-molecule activator of glutamate transporter EAAT2 translation provides neuroprotection.

Glial glutamate transporter EAAT2 plays a major role in glutamate clearance in synaptic clefts. Several lines of evidence indicate that strategies designed to increase EAAT2 expression have potential for preventing excitotoxicity, which contributes to neuronal injury and death in neurodegenerative diseases. We previously discovered several classes of compounds that can increase EAAT2 expression through translational activation. Here, we present efficacy studies of the compound LDN/OSU-0212320, which is a pyridazine derivative from one of our lead series. In a murine model, LDN/OSU-0212320 had good potency, adequate pharmacokinetic properties, no observed toxicity at the doses examined, and low side effect/toxicity potential. Additionally, LDN/OSU-0212320 protected cultured neurons from glutamate-mediated excitotoxic injury and death via EAAT2 activation. Importantly, LDN/OSU-0212320 markedly delayed motor function decline and extended lifespan in an animal model of amyotrophic lateral sclerosis (ALS). We also found that LDN/OSU-0212320 substantially reduced mortality, neuronal death, and spontaneous recurrent seizures in a pilocarpine-induced temporal lobe epilepsy model. Moreover, our study demonstrated that LDN/OSU-0212320 treatment results in activation of PKC and subsequent Y-box-binding protein 1 (YB-1) activation, which regulates activation of EAAT2 translation. Our data indicate that the use of small molecules to enhance EAAT2 translation may be a therapeutic strategy for the treatment of neurodegenerative diseases.

A high-throughput screening method for small-molecule inhibitors of the aberrant mutant SOD1 and dynein complex interaction.

Aberrant protein-protein interactions are attractive drug targets in a variety of neurodegenerative diseases due to the common pathology of accumulation of protein aggregates. In amyotrophic lateral sclerosis, mutations in SOD1 cause the formation of aggregates and inclusions that may sequester other proteins and disrupt cellular processes. It has been demonstrated that mutant SOD1, but not wild-type SOD1, interacts with the axonal transport motor dynein and that this interaction contributes to motor neuron cell death, suggesting that disrupting this interaction may be a potential therapeutic target. However, it can be challenging to configure a high-throughput screening (HTS)-compatible assay to detect inhibitors of a protein-protein interaction. Here we describe the development and challenges of an HTS for small-molecule inhibitors of the mutant SOD1-dynein interaction. We demonstrate that the interaction can be formed by coexpressing the A4V mutant SOD1 and dynein intermediate complex in cells and that this interaction can be disrupted by compounds added to the cell lysates. Finally, we show that some of the compounds identified from a pilot screen to inhibit the protein-protein interaction with this method specifically disrupt the interaction between the dynein complex and mtSOD1 but not the dynein complex itself when applied to live cells.

Lowering of amyloid beta peptide production with a small molecule inhibitor of amyloid-ß precursor protein dimerization.

The amyloid ß precursor protein (APP) is a single-pass transmembrane glycoprotein that is ubiquitously expressed in many cell types, including neurons. Amyloidogenic processing of APP by ß- and γ-secretases leads to the production of amyloid-ß (Aß) peptides that can oligomerize and aggregate into amyloid plaques, a characteristic hallmark of Alzheimer's disease (AD) brains. Multiple reports suggest that dimerization of APP may play a role in Aß production; however, it is not yet clear whether APP dimers increase or decrease Aß and the mechanism is not fully understood. To better understand the relationship between APP dimerization and production of Aß, a high throughput screen for small molecule modulators of APP dimerization was conducted using APP-Firefly luciferase enzyme complementation to detect APP dimerization. Selected modulators identified from a compound library of 77,440 compounds were tested for their effects on Aß generation. Two molecules that inhibited APP dimerization produced a reduction in Aß levels as measured by ELISA. The inhibitors did not change sAPPα or γ-CTF levels, but lowered sAPPß levels, suggesting that blocking the dimerization is preventing the cleavage by ß-secretase in the amyloidogenic processing of APP. To our knowledge, this is the first High Throughput Screen (HTS) effort to identify small molecule modulators of APP dimerization. Inhibition of APP dimerization has previously been suggested as a therapeutic target in AD. The findings reported here further support that modulation of APP dimerization may be a viable means of reducing the production of Aß.

Identification of translational activators of glial glutamate transporter EAAT2 through cell-based high-throughput screening: an approach to prevent excitotoxicity.

Excitotoxicity has been implicated as the mechanism of neuronal damage resulting from acute insults such as stroke, epilepsy, and trauma, as well as during the progression of adult-onset neurodegenerative disorders such as Alzheimer's disease and amyotrophic lateral sclerosis (ALS). Excitotoxicity is defined as excessive exposure to the neurotransmitter glutamate or overstimulation of its membrane receptors, leading to neuronal injury or death. One potential approach to protect against excitotoxic neuronal damage is enhanced glutamate reuptake. The glial glutamate transporter EAAT2 is the quantitatively dominant glutamate transporter and plays a major role in clearance of glutamate. Expression of EAAT2 protein is highly regulated at the translational level. In an effort to identify compounds that can induce translation of EAAT2 transcripts, a cell-based enzyme-linked immunosorbent assay was developed using a primary astrocyte line stably transfected with a vector designed to identify modulators of EAAT2 translation. This assay was optimized for high-throughput screening, and a library of approximately 140,000 compounds was tested. In the initial screen, 293 compounds were identified as hits. These 293 hits were retested at 3 concentrations, and a total of 61 compounds showed a dose-dependent increase in EAAT2 protein levels. Selected compounds were tested in full 12-point dose-response experiments in the screening assay to assess potency as well as confirmed by Western blot, immunohistochemistry, and glutamate uptake assays to evaluate the localization and function of the elevated EAAT2 protein. These hits provide excellent starting points for developing therapeutic agents to prevent excitotoxicity.