Regulation of Proteins Implicated in Alzheimer’s Disease by MicroRNAs

poster abstractAlzheimer’s Disease (AD) is a neurodegenerative disorder characterized by the deposition of Amyloid-Beta (Aβ) peptide in the brain. This toxic peptide is generated by the sequential cleavage of Amyloid Precursor Protein (APP) by Beta-site APP-cleaving enzyme-1 (BACE-1) and γ-secretase. The disorder is also characterized by the perturbation of calcium homeostasis in neurons. MicroRNAs are short, single-stranded RNAs that are able to influence protein expression by targeting the 3’ Untranslated region (UTR) or 5’ UTR of mRNAs. Previous work in our laboratory has shown that miR-101, miR-153 and miR-346 can regulate APP whereas miR-339-5p can lower BACE1 expression. Here, we aim to reduce APP, BACE1 and Aβ levels, in vitro, by the addition of microRNAs that target the 3’ UTR of APP and BACE1. We show that in a human astrocytoma-glioblastoma (U373) cell line, the expression of BACE1 protein is significantly reduced compared to the mock condition upon transfecting miR-298, miR-328 and miR-144. miR-298 also reduces Aβ levels in these cells. Similarly, in HeLa cells, we show that miR-520c, miR-20b and miR-144 produce a reduction in APP expression compared to both mock and a negative control microRNA mimic. Additionally, we observed that knocking down APP using siRNA, but not knocking down BACE1, lowers basal intracellular calcium levels as well as changes the kinetics of Potassium Chloride (KCl)-induced intracellular calcium influx in a human fetal brain (HFB) culture, when compared to control. miR-346 increases basal calcium levels, but does not affect KCl-induced calcium transients in our HFB culture. Taken together, these results show that miRNAs can influence both the protein expression as well as calcium homeostasis in different human cell culture models. By reducing levels of proteins implicated in AD pathology and by reversing calcium dysregulation, our results will benefit AD research and generate possibilities for novel therapeutics

Intravenous immunoglobulin (IVIG) treatment exerts antioxidant and neuropreservatory effects in preclinical models of Alzheimer's disease

Intravenous immunoglobulin (IVIG) has shown limited promise so far in human clinical studies on Alzheimer's disease (AD), yet overwhelmingly positive preclinical work in animals and human brain cultures support the notion that the therapy remains potentially efficacious. Here, we elaborate on IVIG neuropreservation by demonstrating that IVIG protects human primary neurons against oxidative stress in vitro and that IVIG preserves antioxidant defense mechanisms in vivo. Based on these results, we propose the following translational impact: If the dosage and treatment conditions are adequately optimized, then IVIG treatment could play a significant role in preventing and/or delaying the progression of neurodegenerative diseases, such as AD. We suggest that IVIG warrants further investigation to fully exploit its potential as an anti-oxidant, neuroprotective and synapto-protecting agent

Rivastigmine Modifies the α-Secretase Pathway and Potentially Early Alzheimer\u27s Disease

Rivastigmine (or Exelon) is a cholinesterase inhibitor, currently used as a symptomatic treatment for mild-to-moderate Alzheimer’s disease (AD). Amyloid-β peptide (Aβ) generated from its precursor protein (APP) by β-secretase (or BACE1) and γ-secretase endoproteolysis. Alternative APP cleavage by α-secretase (a family of membrane-bound metalloproteases– Adamalysins) precludes the generation of toxic Aβ and yields a neuroprotective and neurotrophic secreted sAPPα fragment. Several signal transduction pathways, including protein kinase C and MAP kinase, stimulate α-secretase. We present data to suggest that rivastigmine, in addition to anticholinesterase activity, directs APP processing away from BACE1 and towards α-secretases. We treated rat neuronal PC12 cells and primary human brain (PHB) cultures with rivastigmine and the α-secretase inhibitor TAPI and assayed for levels of APP processing products and α-secretases. We subsequently treated 3×Tg (transgenic) mice with rivastigmine and harvested hippocampi to assay for levels of APP processing products. We also assayed postmortem human control, AD, and AD brains from subjects treated with rivastigmine for levels of APP metabolites. Rivastigmine dose-dependently promoted α-secretase activity by upregulating levels of ADAM-9, -10, and -17 α-secretases in PHB cultures. Co-treatment with TAPI eliminated rivastigmine-induced sAPPα elevation. Rivastigmine treatment elevated levels of sAPPα in 3×Tg mice. Consistent with these results, we also found elevated sAPPα in postmortem brain samples from AD patients treated with rivastigmine. Rivastigmine can modify the levels of several shedding proteins and directs APP processing toward the non-amyloidogenic pathway. This novel property of rivastigmine can be therapeutically exploited for disease-modifying intervention that goes beyond symptomatic treatment for AD

Initial analysis of peripheral lymphocytic extracellular signal related kinase activation in autism

BACKGROUND: Dysregulation of extracellular signal-related kinase (ERK) activity has been potentially implicated in the pathophysiology of autistic disorder (autism). ERK is part of a central intracellular signaling cascade responsible for a myriad of cellular functions. ERK is expressed in peripheral blood lymphocytes, and measurement of activated (phosphorylated) lymphocytic ERK is commonly executed in many areas of medicine. We sought to conduct the first study of ERK activation in humans with autism by utilizing a lymphocytic ERK activation assay. We hypothesized that ERK activation would be enhanced in peripheral blood lymphocytes from persons with autism compared to those of neurotypical control subjects. METHOD: We conducted an initial study of peripheral lymphocyte ERK activation in 45 subjects with autism and 26 age- and gender-matched control subjects (total n = 71). ERK activation was measured using a lymphocyte counting method (primary outcome expressed as lymphocytes staining positive for cytosolic phosphorylated ERK divided by total cells counted) and additional Western blot analysis of whole cell phosphorylated ERK adjusted for total ERK present in the lymphocyte lysate sample. RESULTS: Cytosolic/nuclear localization of pERK activated cells were increased by almost two-fold in the autism subject group compared to matched neurotypical control subjects (cell count ratio of 0.064 ± 0.044 versus 0.034 ± 0.031; p = 0.002). Elevated phosphorylated ERK levels in whole cell lysates also showed increased activated ERK in the autism group compared to controls (n = 54 total) in Western blot analysis. CONCLUSIONS: The results of this first in human ERK activation study are consistent with enhanced peripheral lymphocytic ERK activation in autism, as well as suggesting that cellular compartmentalization of activated ERK may be altered in this disorder. Future work will be required to explore the impact of concomitant medication use and other subject characteristics such as level of cognitive functioning on ERK activation

Impact of acamprosate on plasma amyloid-β precursor protein in youth: a pilot analysis in fragile X syndrome-associated and idiopathic autism spectrum disorder suggests a pharmacodynamic protein marker

BACKGROUND: Understanding of the pathophysiology of autism spectrum disorder (ASD) remains limited. Brain overgrowth has been hypothesized to be associated with the development of ASD. A derivative of amyloid-β precursor protein (APP), secreted APPα (sAPPα), has neuroproliferative effects and has been shown to be elevated in the plasma of persons with ASD compared to control subjects. Reduction in sAPPα holds promise as a novel molecular target of treatment in ASD. Research into the neurochemistry of ASD has repeatedly implicated excessive glutamatergic and deficient GABAergic neurotransmission in the disorder. With this in mind, acamprosate, a novel modulator of glutamate and GABA function, has been studied in ASD. No data is available on the impact of glutamate or GABA modulation on sAPPα function. METHODS: Plasma APP derivative levels pre- and post-treatment with acamprosate were determined in two pilot studies involving youth with idiopathic and fragile X syndrome (FXS)-associated ASD. We additionally compared baseline APP derivative levels between youth with FXS-associated or idiopathic ASD. RESULTS: Acamprosate use was associated with a significant reduction in plasma sAPP(total) and sAPPα levels but no change occurred in Aβ40 or Aβ42 levels in 15 youth with ASD (mean age: 11.1 years). Youth with FXS-associated ASD (n = 12) showed increased sAPPα processing compared to age-, gender- and IQ-match youth with idiopathic ASD (n = 11). CONCLUSIONS: Plasma APP derivative analysis holds promise as a potential biomarker for use in ASD targeted treatment. Reduction in sAPP (total) and sAPPα may be a novel pharmacodynamic property of acamprosate. Future study is required to address limitations of the current study to determine if baseline APP derivative analysis may predict subgroups of persons with idiopathic or FXS-associated ASD who may respond best to acamprosate or to potentially other modulators of glutamate and/or GABA neurotransmission

Synthesis of the Alzheimer drug Posiphen into its primary metabolic products (+)-N1-norPosiphen, (+)-N8-norPosiphen and (+)-N1, N8-bisnorPosiphen, their inhibition of amyloid precursor protein, α-Synuclein synthesis, interleukin-1β release, and cholinergic action

A major pathological hallmark of Alzheimer disease (AD) is the appearance in the brain of senile plaques that are primarily composed of aggregated forms of β-amyloid peptide (Aβ) that derive from amyloid precursor protein (APP). Posiphen (1) tartrate is an experimental AD drug in current clinical trials that reduces Aβ levels by lowering the rate of APP synthesis without toxicity. To support the clinical development of Posiphen (1) and elucidate its efficacy, its three major metabolic products, (+)-N1-norPosiphen (15), (+)-N8-norPosiphen (17) and (+)-N1, N8-bisnorPosiphen (11), were required in high chemical and optical purity. The efficient transformation of Posiphen (1) into these metabolic products, 15, 17 and 11, is described. The biological activity of these metabolites together with Posiphen (1) and its enantiomer, the AD drug candidate (-)-phenserine (2), was assessed against APP,α-synuclein and classical cholinergic targets. All the compounds potently inhibited the generation of APP and α-synuclein in neuronal cultures. In contrast, metabolites 11 and 15, and (-)-phenserine (2) but not Posiphen (1) or 17, possessed acetyl cholinesterase inhibitory action and no compounds bound either nicotinic or muscarinic receptors. As Posiphen (1) lowered CSF markers of inflammation in a recent clinical trial, the actions of 1 and 2 on proinflammatory cytokine interleukin (IL)-1β release human peripheral blood mononuclear cells was evaluated, and found to be potently inhibited by both agents

Rivastigmine modifies the α-secretase pathway and potentially early Alzheimer's disease

Rivastigmine (or Exelon) is a cholinesterase inhibitor, currently used as a symptomatic treatment for mild-to-moderate Alzheimer’s disease (AD). Amyloid-β peptide (Aβ) generated from its precursor protein (APP) by β-secretase (or BACE1) and γ-secretase endoproteolysis. Alternative APP cleavage by α-secretase (a family of membrane-bound metalloproteases– Adamalysins) precludes the generation of toxic Aβ and yields a neuroprotective and neurotrophic secreted sAPPα fragment. Several signal transduction pathways, including protein kinase C and MAP kinase, stimulate α-secretase. We present data to suggest that rivastigmine, in addition to anticholinesterase activity, directs APP processing away from BACE1 and towards α-secretases. We treated rat neuronal PC12 cells and primary human brain (PHB) cultures with rivastigmine and the α-secretase inhibitor TAPI and assayed for levels of APP processing products and α-secretases. We subsequently treated 3×Tg (transgenic) mice with rivastigmine and harvested hippocampi to assay for levels of APP processing products. We also assayed postmortem human control, AD, and AD brains from subjects treated with rivastigmine for levels of APP metabolites. Rivastigmine dose-dependently promoted α-secretase activity by upregulating levels of ADAM-9, -10, and -17 α-secretases in PHB cultures. Co-treatment with TAPI eliminated rivastigmine-induced sAPPα elevation. Rivastigmine treatment elevated levels of sAPPα in 3×Tg mice. Consistent with these results, we also found elevated sAPPα in postmortem brain samples from AD patients treated with rivastigmine. Rivastigmine can modify the levels of several shedding proteins and directs APP processing toward the non-amyloidogenic pathway. This novel property of rivastigmine can be therapeutically exploited for disease-modifying intervention that goes beyond symptomatic treatment for AD

Rivastigmine Lowers Aβ and Increases sAPPα Levels, Which Parallel Elevated Synaptic Markers and Metabolic Activity in Degenerating Primary Rat Neurons

Overproduction of amyloid-β (Aβ) protein in the brain has been hypothesized as the primary toxic insult that, via numerous mechanisms, produces cognitive deficits in Alzheimer's disease (AD). Cholinesterase inhibition is a primary strategy for treatment of AD, and specific compounds of this class have previously been demonstrated to influence Aβ precursor protein (APP) processing and Aβ production. However, little information is available on the effects of rivastigmine, a dual acetylcholinesterase and butyrylcholinesterase inhibitor, on APP processing. As this drug is currently used to treat AD, characterization of its various activities is important to optimize its clinical utility. We have previously shown that rivastigmine can preserve or enhance neuronal and synaptic terminal markers in degenerating primary embryonic cerebrocortical cultures. Given previous reports on the effects of APP and Aβ on synapses, regulation of APP processing represents a plausible mechanism for the synaptic effects of rivastigmine. To test this hypothesis, we treated degenerating primary cultures with rivastigmine and measured secreted APP (sAPP) and Aβ. Rivastigmine treatment increased metabolic activity in these cultured cells, and elevated APP secretion. Analysis of the two major forms of APP secreted by these cultures, attributed to neurons or glia based on molecular weight showed that rivastigmine treatment significantly increased neuronal relative to glial secreted APP. Furthermore, rivastigmine treatment increased α-secretase cleaved sAPPα and decreased Aβ secretion, suggesting a therapeutic mechanism wherein rivastigmine alters the relative activities of the secretase pathways. Assessment of sAPP levels in rodent CSF following once daily rivastigmine administration for 21 days confirmed that elevated levels of APP in cell culture translated in vivo. Taken together, rivastigmine treatment enhances neuronal sAPP and shifts APP processing toward the α-secretase pathway in degenerating neuronal cultures, which mirrors the trend of synaptic proteins, and metabolic activity

Increased Secreted Amyloid Precursor Protein-α (sAPPα) in Severe Autism: Proposal of a Specific, Anabolic Pathway and Putative Biomarker

Autism is a neurodevelopmental disorder characterized by deficits in verbal communication, social interactions, and the presence of repetitive, stereotyped and compulsive behaviors. Excessive early brain growth is found commonly in some patients and may contribute to disease phenotype. Reports of increased levels of brain-derived neurotrophic factor (BDNF) and other neurotrophic-like factors in autistic neonates suggest that enhanced anabolic activity in CNS mediates this overgrowth effect. We have shown previously that in a subset of patients with severe autism and aggression, plasma levels of the secreted amyloid-β (Aβ) precursor protein-alpha form (sAPPα) were significantly elevated relative to controls and patients with mild-to-moderate autism. Here we further tested the hypothesis that levels of sAPPα and sAPPβ (proteolytic cleavage products of APP by α- and β-secretase, respectively) are deranged in autism and may contribute to an anabolic environment leading to brain overgrowth. We measured plasma levels of sAPPα, sAPPβ, Aβ peptides and BDNF by corresponding ELISA in a well characterized set of subjects. We included for analysis 18 control, 6 mild-to-moderate, and 15 severely autistic patient plasma samples. We have observed that sAPPα levels are increased and BDNF levels decreased in the plasma of patients with severe autism as compared to controls. Further, we show that Aβ1-40, Aβ1-42, and sAPPβ levels are significantly decreased in the plasma of patients with severe autism. These findings do not extend to patients with mild-to-moderate autism, providing a biochemical correlate of phenotypic severity. Taken together, this study provides evidence that sAPPα levels are generally elevated in severe autism and suggests that these patients may have aberrant non-amyloidogenic processing of APP

Exendin-4 Ameliorates Motor Neuron Degeneration in Cellular and Animal Models of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by a progressive loss of lower motor neurons in the spinal cord. The incretin hormone, glucagon-like peptide-1 (GLP-1), facilitates insulin signaling, and the long acting GLP-1 receptor agonist exendin-4 (Ex-4) is currently used as an anti-diabetic drug. GLP-1 receptors are widely expressed in the brain and spinal cord, and our prior studies have shown that Ex-4 is neuroprotective in several neurodegenerative disease rodent models, including stroke, Parkinson's disease and Alzheimer's disease. Here we hypothesized that Ex-4 may provide neuroprotective activity in ALS, and hence characterized Ex-4 actions in both cell culture (NSC-19 neuroblastoma cells) and in vivo (SOD1 G93A mutant mice) models of ALS. Ex-4 proved to be neurotrophic in NSC-19 cells, elevating choline acetyltransferase (ChAT) activity, as well as neuroprotective, protecting cells from hydrogen peroxide-induced oxidative stress and staurosporine-induced apoptosis. Additionally, in both wild-type SOD1 and mutant SOD1 (G37R) stably transfected NSC-19 cell lines, Ex-4 protected against trophic factor withdrawal-induced toxicity. To assess in vivo translation, SOD1 mutant mice were administered vehicle or Ex-4 at 6-weeks of age onwards to end-stage disease via subcutaneous osmotic pump to provide steady-state infusion. ALS mice treated with Ex-4 showed improved glucose tolerance and normalization of behavior, as assessed by running wheel, compared to control ALS mice. Furthermore, Ex-4 treatment attenuated neuronal cell death in the lumbar spinal cord; immunohistochemical analysis demonstrated the rescue of neuronal markers, such as ChAT, associated with motor neurons. Together, our results suggest that GLP-1 receptor agonists warrant further evaluation to assess whether their neuroprotective potential is of therapeutic relevance in ALS