Debomoy K. Lahiri

GLP-1 receptor stimulation preserves primary cortical and dopaminergic neurons in cellular and rodent models of stroke and Parkinsonism

Glucagon-like peptide-1 (GLP-1) is an endogenous insulinotropic peptide secreted from the gastrointestinal tract in response to food intake. It enhances pancreatic islet β-cell proliferation and glucose-dependent insulin secretion, and lowers blood glucose and food intake in patients with type 2 diabetes mellitus (T2DM). A long-acting GLP-1 receptor (GLP-1R) agonist, exendin-4 (Ex-4), is the first of this new class of antihyperglycemia drugs approved to treat T2DM. GLP-1Rs are coupled to the cAMP second messenger pathway and, along with pancreatic cells, are expressed within the nervous system of rodents and humans, where receptor activation elicits neurotrophic actions. We detected GLP-1R mRNA expression in both cultured embryonic primary cerebral cortical and ventral mesencephalic (dopaminergic) neurons. These cells are vulnerable to hypoxia- and 6-hydroxydopamine–induced cell death, respectively. We found that GLP-1 and Ex-4 conferred protection in these cells, but not in cells from Glp1r knockout (-/-) mice. Administration of Ex-4 reduced brain damage and improved functional outcome in a transient middle cerebral artery occlusion stroke model. Ex-4 treatment also protected dopaminergic neurons against degeneration, preserved dopamine levels, and improved motor function in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinsons disease (PD). Our findings demonstrate that Ex-4 can protect neurons against metabolic and oxidative insults, and they provide preclinical support for the therapeutic potential for Ex-4 in the treatment of stroke and PD.

Alzheimer's Disease (AD)-Like Pathology in Aged Monkeys after Infantile Exposure to Environmental Metal Lead (Pb): Evidence for a Developmental Origin and Environmental Link for AD

The sporadic nature of Alzheimers disease (AD) argues for an environmental link that may drive AD pathogenesis; however, the triggering factors and the period of their action are unknown. Recent studies in rodents have shown that exposure to lead (Pb) during brain development predetermined the expression and regulation of the amyloid precursor protein (APP) and its amyloidogenic β-amyloid (Aβ) product in old age. Here, we report that the expression of AD-related genes [APP, BACE1 (β-site APP cleaving enzyme 1)] as well as their transcriptional regulator (Sp1) were elevated in aged (23-year-old) monkeys exposed to Pb as infants. Furthermore, developmental exposure to Pb altered the levels, characteristics, and intracellular distribution of Aβ staining and amyloid plaques in the frontal association cortex. These latent effects were accompanied by a decrease in DNA methyltransferase activity and higher levels of oxidative damage to DNA, indicating that epigenetic imprinting in early life influenced the expression of AD-related genes and promoted DNA damage and pathogenesis. These data suggest that AD pathogenesis is influenced by early life exposures and argue for both an environmental trigger and a developmental origin of AD.

Glucagon-like peptide-1 decreases endogenous amyloid-β peptide (Aβ) levels and protects hippocampal neurons from death induced by Aβ and iron

Glucagon‐like peptide‐1(7–36)‐amide (GLP‐1) is an endogenous insulinotropic peptide that is secreted from the gastrointestinal tract in response to food. It enhances pancreatic islet β‐cell proliferation and glucose‐dependent insulin secretion and lowers blood glucose and food intake in patients with type 2 diabetes mellitus. GLP‐1 receptors, which are coupled to the cyclic AMP second messenger pathway, are expressed throughout the brains of rodents and humans. It was recently reported that GLP‐1 and exendin‐4, a naturally occurring, more stable analogue of GLP‐1 that binds at the GLP‐1 receptor, possess neurotrophic properties and can protect neurons against glutamate‐induced apoptosis. We report here that GLP‐1 can reduce the levels of amyloid‐β peptide (Aβ) in the brain in vivo and can reduce levels of amyloid precursor protein (APP) in cultured neuronal cells. Moreover, GLP‐1 and exendin‐4 protect cultured hippocampal neurons against death induced by Aβ and iron, an oxidative insult. Collectively, these data suggest that GLP‐1 can modify APP processing and protect against oxidative injury, two actions that suggest a novel therapeutic target for intervention in Alzheimers disease.

Epigenetics, oxidative stress, and Alzheimer disease.

Alzheimer disease (AD) is a progressive neurodegenerative disorder whose clinical manifestations appear in old age. The sporadic nature of 90% of AD cases, the differential susceptibility to and course of the illness, as well as the late age onset of the disease suggest that epigenetic and environmental components play a role in the etiology of late-onset AD. Animal exposure studies demonstrated that AD may begin early in life and may involve an interplay between the environment, epigenetics, and oxidative stress. Early life exposure of rodents and primates to the xenobiotic metal lead (Pb) enhanced the expression of genes associated with AD, repressed the expression of others, and increased the burden of oxidative DNA damage in the aged brain. Epigenetic mechanisms that control gene expression and promote the accumulation of oxidative DNA damage are mediated through alterations in the methylation or oxidation of CpG dinucleotides. We found that environmental influences occurring during brain development inhibit DNA-methyltransferases, thus hypomethylating promoters of genes associated with AD such as the beta-amyloid precursor protein (APP). This early life imprint was sustained and triggered later in life to increase the levels of APP and amyloid-beta (Abeta). Increased Abeta levels promoted the production of reactive oxygen species, which damage DNA and accelerate neurodegenerative events. Whereas AD-associated genes were overexpressed late in life, others were repressed, suggesting that these early life perturbations result in hypomethylation as well as hypermethylation of genes. The hypermethylated genes are rendered susceptible to Abeta-enhanced oxidative DNA damage because methylcytosines restrict repair of adjacent hydroxyguanosines. Although the conditions leading to early life hypo- or hypermethylation of specific genes are not known, these changes can have an impact on gene expression and imprint susceptibility to oxidative DNA damage in the aged brain.

The Fetal Basis of Amyloidogenesis: Exposure to Lead and Latent Overexpression of Amyloid Precursor Protein and β-Amyloid in the Aging Brain

The fetal basis of adult disease (FeBAD) hypothesis states that many adult diseases have a fetal origin. According to FeBAD, injury or environmental influences occurring at critical periods of organ development could result in “programmatic” changes via alterations in gene expression or gene imprinting that may result in functional deficits that become apparent later in life. Alzheimers disease (AD) is a progressive neurodegenerative disorder that is characterized by excessive deposits of aggregated β-amyloid (Aβ) peptides, which are snippets of the β-amyloid precursor protein (APP). The predominately sporadic nature of AD suggests that the environment must play a role in neurodegeneration. To examine latent responses to an environmental agent, we exposed rodents to lead and monitored the lifetime expression of the APP gene. We observed that APP mRNA expression was transiently induced in neonates, but exhibited a delayed overexpression 20 months after exposure to Pb had ceased. This upregulation in APP mRNA expression was commensurate with a rise in activity of the transcription factor Sp1, one of the regulators of the APP gene. Furthermore, the increase in APP gene expression in old age was accompanied by an elevation in APP and its amyloidogenic Aβ product. In contrast, APP expression, Sp1 activity, as well as APP and Aβ protein levels were unresponsive to Pb exposure during old age. These data suggested that environmental influences occurring during brain development predetermined the expression and regulation of APP later in life, potentially altering the course of amyloidogenesis.

A New Therapeutic Target in Alzheimer's Disease Treatment: Attention to Butyrylcholinesterase

Summary Alzheimers disease (AD) is a progressive neurodegenerative disorder of the elderly, characterised by widespread loss central cholinergic function. The only symptomatic treatment proven effective, to date is the use of cholinesterase (ChE) inhibitors to augment surviving cholinergic activity. ChE inhibitors act on the enzymes that hydrolyse acetylcholine (ACh) following synaptic release. In the healthy brain, acetylcholinesterase (AChE) predominates (80%) and butyrylcholinesterase (BuChE) is considered to play a minor role in regulating brain ACh levels. In the AD brain, BuChE activity rises while AChE activity remains unchanged or declines. Therefore both enzymes are likely to have involvement in regulating ACh levels and represent legitimate therapeutic targets to ameliorate the cholinergic deficit. The two enzymes differ in location, substrate specificity and kinetics. Recent evidence suggests that BuChE may also have a role in the aetiology and progression of AD beyond regulation of synaptic ACh levels. Experimental evidence from the use of agents with enhanced selectivity for BuChE (cymserine, MF-8622) and ChE inhibitors such as rivastigmine, which have a dual inhibitory action on both AChE and BuChE, indicate potential therapeutic benefits of inhibiting both AChE and BuChE in AD and related dementias. The development of specific BuChE inhibitors and the continued use of ChE inhibitors with the ability to inhibit BuChE in addition to AChE should lead to improved clinical outcomes.

DNA isolation by a rapid method from human blood samples: Effects of MgCl2, EDTA, storage time, and temperature on DNA yield and quality

The isolation of DNA from whole blood by a modified rapid method (RM) was tested using various detergents and buffer conditions. Extraction of DNA with either NP-40 or Triton X-100 gave a high yield of undegraded DNA in less than an hour. The concentration of magnesium ion in the buffers was critical to obtaining intact, high molecular weight (HMW) DNA. Greater than 10 mM MgCl2 led to degradation. Addition of EDTA to the buffer inhibits this degradation. Preparation of DNA from blood stored at room temperature or incubated at 37°C for 24 hr resulted in the same amount and quality of DNA as from samples frozen at −70°C. DNA from blood samples that had undergone more than four freeze-thaw cycles was found to be partially degraded. The modified RM can be applied to extract DNA from as little as 10 µl of blood (340 ng of DNA) and from dried blood samples. DNA samples remained intact and undegraded for longer times when DNA was dissolved in higher concentrations of EDTA.

GLP-1 Receptor Stimulation Reduces Amyloid-β Peptide Accumulation and Cytotoxicity in Cellular and Animal Models of Alzheimer’s Disease

Type 2 (T2) diabetes mellitus (DM) has been associated with an increased incidence of neurodegenerative disorders, including Alzheimers disease (AD). Several pathological features are shared between diabetes and AD, including dysfunctional insulin signaling and a dysregulation of glucose metabolism. It has therefore been suggested that not only may the two conditions share specific molecular mechanisms but also that agents with proven efficacy in one may be useful against the other. Hence, the present study characterized the effects of a clinically approved long-acting analogue, exendin-4 (Ex-4), of the endogenous insulin releasing incretin, glucagon-like peptide-1 (GLP-1), on stress-induced toxicity in neuronal cultures and on amyloid-beta protein (Abeta) and tau levels in triple transgenic AD (3xTg-AD) mice with and without streptozocin (STZ)-induced diabetes. Ex-4 ameliorated the toxicity of Abeta and oxidative challenge in primary neuronal cultures and human SH-SY5Y cells in a concentration-dependent manner. When 11 to 12.5 month old female 3xTg AD mice were challenged with STZ or saline, and thereafter treated with a continuous subcutaneous infusion of Ex-4 or vehicle, Ex-4 ameliorated the diabetic effects of STZ in 3xTg-AD mice, elevating plasma insulin and lowering both plasma glucose and hemoglobin A1c (HbA1c) levels. Furthermore, brain levels of Abeta protein precursor and Abeta, which were elevated in STZ 3xTg-AD mice, were significantly reduced in Ex-4 treated mice. Brain tau levels were unaffected following STZ challenge, but showed a trend toward elevation that was absent following Ex-4 treatment. Together, these results suggest a potential value of Ex-4 in AD, particularly when associated with T2DM or glucose intolerance.

Butyrylcholinesterase: An Important New Target in Alzheimer's Disease Therapy

Acetylcholinesterase (AChE) predominates in the healthy brain, with butyrylcholinesterase (BuChE) considered to play a minor role in regulating brain acetylcholine (ACh) levels. However, BuChE activity progressively increases in patients with Alzheimers disease (AD), while AChE activity remains unchanged or declines. Both enzymes therefore represent legitimate therapeutic targets for ameliorating the cholinergic deficit considered to be responsible for the declines in cognitive, behavioral and global functioning characteristic of AD. The two enzymes differ in substrate specificity, kinetics and activity in different brain regions. Experimental evidence from the use of agents with enhanced selectivity for BuChE (cymserine analogues, MF-8622) and the dual inhibitor of both AChE and BuChE, rivastigmine, indicates potential therapeutic benefits of inhibiting both AChE and BuChE in AD and related dementias. Recent evidence suggests that both AChE and BuChE may have roles in the aetiology and progression of AD beyond regulation of synaptic ACh levels. The development of specific BuChE inhibitors and further experience with the dual enzyme inhibitor rivastigmine will improve understanding of the aetiology of AD and should lead to a wider variety of potent treatment options.

Treatment outcome of tacrine therapy depends on apolipoprotein genotype and gender of the subjects with Alzheimer's disease

We studied the effects of apolipoprotein E (APOE) genotype and gender on clinical response to tacrine in patients with mild to moderate Alzheimers disease (AD). We analyzed data from a previously reported 30-week, double-blind, placebo-controlled trial of tacrine, in which APOE genotypes were determined from previously collected plasma samples. Patients were assigned to placebo or tacrine with daily dosages of 80, 120, or 160 mg/day. The outcome measures were Alzheimers Disease Assessment Scale-Cognitive Component, Clinician Interview Based Impression, Mini-Mental State Examination, and the Caregiver-rated Clinical Global Impression of Change. An intent-to-treat (ITT) analysis of patients with available genotypes (n = 528) did not reveal response differences by genotype, although the effect size was twice as large in the ϵ2-3 as theϵ4 group (-2.62 versus -1.25). The association of treatment effect with APOE genotype varied significantly according to gender (p< 0.002 for ITT; p < 0.05 for evaluables). The treatment effect was larger in the ϵ2-3 compared with ϵ4 women (ITT, 4.24 points, p = 0.03; evaluable, 7.20 points, p = 0.01). In contrast, treatment effect size was not different between ϵ2-3 andϵ4 of men with AD. APOE genotype and gender may predict response to tacrine in patients with AD.