Kelly T. Dineley, Ph.D. Associate Professor

 

Research Interests:

Alzheimer’s Disease

Elevation of beta-amyloid peptides (Aβ) is considered a culprit in the cognitive decline of Alzheimer’s disease (AD), though these peptides are present in the brains and CSF of normal subjects.   The earliest detected cognitive dysfunction in AD occurs in the hippocampus: impairment in the encoding of new episodic memories is typical of early AD and this loss of episodic memory is linked to medial temporal pathology including the hippocampus.  Despite intensive study, the mechanism by which elevated Aβ leads to AD-related hippocampal dysfunction remains mysterious, not to mention the lack of an understanding of the physiologic role for Aβ in normal cognition, synaptic function, and signal transduction.   

Nicotinic Receptors in AD

An early depletion of nicotinic acetylcholine receptors (nAChRs) on the cholinergic projection neurons in the brains of AD patients is a universal feature of the disease; these receptors are the target for the current symptomatic strategy.  α7 nAChRs are expressed by these cholinergic projection neurons as well as being expressed pre- and post-synaptically in target brain regions.  α7 nAChRs flux the pluripotent second messenger Ca²⁺ and have been shown to modulate both neuron excitability and plasticity.  Furthermore, α7 nAChRs have been implicated in mediating some of the cognitive enhancing effects of in vivo administered nicotine. 

Originating with the observation of an association between Ab and a7 nAChRs in postmortem tissue from AD brain, we and others have gone on to show that Aβ binds to α7 nAChRs, leads to Ca²⁺-dependent activation of the extracellular signal-regulated kinase mitogen-activated protein kinase (ERK MAPK) cascade that results in phosphorylation of the transcription factor CREB.  The ERK MAPK cascade is crucial for many forms of synaptic plasticity, learning and memory We also demonstrated that Aβ activates α7 nAChRs expressed in Xenopus oocytes, suggesting that Aβ is an endogenous ligand for the α7 nAChR.  In the Tg2576 animal model for AD in which Aβ production is elevated throughout the animals’ lifetime, we have shown age dependent dysregulation of ERK2 MAPK and CREB as well as a dramatic up-regulation of α7 nAChRs in the hippocampi of these mice, indicating that chronic exposure to Aβ perturbs the normal functionality of this receptor system.  Furthermore, Tg2576 mice exhibit an age-dependent decline in cognition. 

This project strives to understand how Ab interaction with a7 nAChRs contributes to the molecular etiology of AD.  Currently, we are investigating whether α7 nAChRs mediate the toxic effects of elevated Aβ by generating and studying genetic animal models in which Aβ is over produced and α7 nAChR function is altered.  These animal models are analyzed at the behavioral, electrophysiological, biochemical and histopathological levels.  This work could potentially provide a mechanism for the cholinergic deficit in AD, as well as help delineate the emerging field of Ab / a7 nAChR biology as it pertains to normal physiological processes.  

Additional investigations into the biological processes underlying cognitive decline in AD focus on the role of calcineurin (CaN) in collaboration with Dr. Giulio Taglialatela, and that of peroxisome proliferator-activated receptor gamma  (PPARγ) in collaboration with Dr. Larry Denner.  Both these projects are currently funded at the RO1 level with the NIA.   

CaN in AD

The Tg2576 transgenic mouse is an extensively characterized animal model for Alzheimer’s disease (AD).  Similar to AD, these mice suffer from progressive decline in several forms of declarative memory including contextual fear conditioning and novel object recognition (NOR).  Recent work on this and other AD animal models suggests that initial cognitive deficits are due to synaptic dysfunction that is triggered by oligomeric forms of Aβ peptide.  These early forms of cognitive dysfunction, we believe, are fully treatable with the correct intervention.   

Tg2576 at an early age have high central nervous system (CNS) activity of calcineurin (CaN), a phosphatase involved in negative regulation of memory function via inactivation of the transcription factor cAMP responsive element binding proteins (CREB), and display CaN-dependent memory deficits. These results thus suggested the involvement of prefibrillary forms of Aβ. To investigate this issue, we compared the effect of monomeric, oligomeric, and fibrillar Aβ on CaN activity, CaN-dependent pCREB and phosphorylated Bcl-2 Associated death Protein (pBAD) levels, and cell death in SY5Y cells and in rat brain slices, and determined the role of CaN on CREB phosphorylation in the CNS of Tg2576 mice. Our results show that oligomeric Aβ specifically induces CaN activity and promotes CaN-dependent CREB and Bcl-2 Asociated death Protein (BAD) dephosphorylation and cell death. Furthermore, Tg2576 mice display Aβ oligomers and reduced pCREB in the CNS, which is normalized by CaN inhibition. These findings suggest a role for CaN in mediating effects of oligomeric Aβ on neural cells. Because elevated CaN levels have been reported in the CNS of cognitively impaired aged rodents, our results further suggest that abnormal CaN hyperactivity may be a common event exacerbating the cognitive and neurodegenerative impact of oligomeric Aβ in the aging CNS.

Further studies have demonstrated that acute CaN inhibition rescues deficits in different forms of declarative memory in 5 months old Tg2576; specifically spontaneous object recognition, by employing the NOR paradigm, and contextual fear conditioning.  Furthermore, we determined whether FK506 rescue of NOR deficits depends on the retention interval employed and therefore is restricted to short-term, intermediate-term, or long-term memory (STM, ITM or LTM, respectively).  In object recognition, Tg2576 are unimpaired when NOR is tested as a STM task and CaN inhibition with FK506 does not influence NOR STM performance in Tg2576 or WT mice.  Tg2576 were impaired in NOR compared to WT mice when a 4 or 24 hour retention interval was employed to model ITM and LTM, respectively.  Acute CaN inhibition prior to and during the training session reversed these deficits in Tg2576 mice with no effect on WT performance. Our findings demonstrate that aberrant CaN activity mediates object recognition deficits in 5 months old Tg2576 when NOR is employed as a test for ITM and LTM.  Additionally, aberrant CaN activity mediates associative learning and memory deficits as well.  In human AD, CaN inhibition may lead the way for therapeutics to improve declarative memory performance as demonstrated in a mouse model for AD.

PPARγ in AD

Converging evidence associates gluco-regulatory abnormalities and cognitive function in AD.  As such, ongoing clinical trials are testing whether insulin-sensitizing drugs, such as the peroxisome-proliferator-activated receptor gamma (PPARγ) agonist, rosiglitazone (RTZ), improve AD-related cognitive performance.  In this study, we used the AD mouse model Tg2576 to test the hypothesis that efficacy of one-month RTZ treatment to improve cognitive performance correlates with peripheral gluco-regulatory status.  We assessed cognition and peripheral gluco-regulatory status of Tg2576 mice following one month treatment with RTZ initiated at 4, 8, and 12 month of age. Our study shows that in 5 MO Tg2576, which are cognitively impaired and with normal glucose regulation, one month treatment with RTZ did not improve cognitive behavior.  In 9 and 13 MO Tg2576, RTZ treatment effectively normalized peripheral gluco-regulatory abnormalities; however, reversal of cognitive deficits only occurred in 9 MO mice.  These findings suggest that RTZ effectiveness at ameliorating cognitive deficits is greater when initiated prior to chronic peripheral gluco-regulatory abnormalities. 

Based on our studies, we propose a model in which RTZ treatment reverses learning and memory deficits that occur concomitant with insulin resistance through activation of CNS PPARγ.  We showed that RTZ rescue of associative learning deficits in Tg2576 was reversed by acute intracerebroventricular (icv) injection of the specific and irreversible PPARγ antagonist, GW9662 (GW; Seimandi et al., 2005), clearly demonstrating the RTZ effect was mediated specifically by the PPARγ isoform in the CNS.  PPARγ specificity was also shown by RTZ-dependent increase in hippocampal PPARγ DNA binding activity which was reversed by acute icv GW, with no effects on the PPARα or PPARδ isoforms.  Regional specificity was observed since these pharmacological manipulations did not alter PPARγ, PPARα, or PPARδ in the cerebellum.  Collectively, these data suggest that RTZ improves cognitive function in hippocampus-dependent cognition specifically through forebrain activation of the PPARγ isoform.  This is the first demonstration that PPARγ plays a role in cognitive function.  As such, little is known about the regulation of the PPARγ signaling axis in brain or the molecular mechanisms mediating RTZ-induced PPARγ activity in the CNS.  Our research plan is focused on defining the CNS PPARγ-specific signaling axis that rescues cognitive deficits in Tg2576.   

Parkinson’s Disease

The cytosolic protein alpha-synuclein (A-SYN) is enriched at the pre-synaptic terminals of almost all types of neurons in the central nervous system. A-SYN overexpression and the expression of three different mutants have been shown to sustain the pathogenesis of selected forms of Parkinson's disease (PD). The localization of the protein and the defects found in knocked out or transgenic animals suggest a role of A-SYN in the regulation of synaptic efficiency.

Alpha-Synuclein Oligomers in PD Pathophysiology

Although the exact function(s) of A-SYN remain uncertain, the preferential localization to presynaptic nerve terminals and its interaction with vesicular phospholipids and proteins suggest a regulatory function associated with synaptic activity, dopamine (DA) production and metabolism, lipid vesicle trafficking, and chaperone-like activity).  However, the precise function of the protein and the molecular mechanisms of its action are still unclear.  Our working hypothesis is that A-SYN oligomer formation (whether intra or extracellular) disrupts synaptic plasticity, learning and memory through disruption of post-synaptic processes.  Significance: Innovation: Conformation- and A-SYN-specific anti-oligomer antibodies as a research tool and possible therapeutic and diagnostic tool 

Addiction

PPARγ as a Therapeutic Target in Drug Abuse

Psychostimulant abuse and vulnerability to dependence (addiction) and relapse during abstinence linger as major public health problems in the United States and effective, safe pharmacotherapeutic approaches are needed. Here, we will explore a novel pharmacological target, peroxisome proliferator-activated receptor (PPAR), using a preclinical model of addiction in vivo. This ligand-activated transcription factor belongs to the nuclear hormone receptor superfamily and its gamma isoform (PPARγ) plays a critical physiological role as a primary lipid sensor and regulator of lipid metabolism.  Thus, there are several FDA-approved ligands that are clinically used for the treatment of some diseases of the periphery, including type 2 diabetes and hyperglycemia.  However, PPARγ is widely distributed in the CNS and is abundantly expressed by neurons; much recent work has demonstrated a protective role for PPARγ in the neuronal plasticity seen in many CNS injury models such as traumatic brain injury and stroke.  While the beneficial effect of PPARγ agonism in these models is mainly attributed to an anti-inflammatory role, we have recently shown that CNS PPARγ rescues hippocampus-dependent cognitive deficits in an animal model of Alzheimer’s disease in a manner that, at least in part, involves recruitment of hippocampal ERK MAPK activity to the nucleus (Rodriguez et al., 2010).  Given the important role for learning and memory in the processes through which drug abuse transitions to addiction,  combined with our recent evidence for a role for CNS PPARγ in restoring cognitive function through effects on ERK MAPK, we hypothesize that that neuronal PPARγ represents a potential therapeutic target in maintaining abstinence from cocaine self administration.  This pilot study has the potential to validate PPARγ agonist as a new therapeutic for maintaining abstinence from cocaine use for which there are currently no available pharmacotherapies.  Because we utilize FDA-approved PPARγ agonists in the present study, the resultant preclinical data will set the stage to expedite clinical research to evaluate efficacy in human cocaine abusers. Thus, this project addresses a fundamental gap in addiction research, namely, the need for medications that effectively suppress relapse to stimulant dependence with a limited side effect profile.  

Cancer

Alcohol‐Nicotine Synergism in Head and Neck Cancer

Head and neck squamous cell carcinoma (HNSCC) is one of the most common neoplasms in the world. HNSCC represents approximately 6% of all new cancers in the US and approximately 350,000 people are diagnosed annually worldwide with HNSCC.  Despite advances in surgical treatment and the institution of novel chemo/radiation approaches, patient prognosis has not improved in more than 30 years, with the 5-year survival remaining at 50% - 60%.  The high rate of local recurrence and considerable mortality that accompany this type of cancer mandate the development of new in vivo and in vitro models to test clinically-significant, mechanistic hypotheses, the outcomes of which will drive the development of novel therapeutic approaches to the management of HNSCC.

The combined exposure to cigarette smoking and alcohol drinking appears to account for 75% of HNSCC suggesting a multiplicative effect of smoking and drinking on carcinogenesis.  Although our understanding of genetic changes in cancer has been forwarded by tremendous investment in research focused at this aspect of disease, little work has focused on understanding the interplay between tobacco and alcohol abuse and the genesis of cancer.

The continuous cycle of birth and death of oral cavity keratinocytes is a self-sustained process controlled, in part, by locally produced acetylcholine.  It is now known that specific subtypes of nicotinic acetylcholine receptors (nAChRs) expressed by oral keratinocytes are responsive to both nicotine and alcohol and that these compounds lead to alterations in nAChR stoichiometry through changes in nAChR subunit expression.  Cell culture studies have shown that nicotine in tobacco products and metabolites of alcohol contribute to squamatization of oral keratinocytes by altering cell cycle control.  As such, chronic tobacco or alcohol abuse has the potential to contribute to squamatization of oral keratinocytes through direct effects on nAChRs.  Whether abuse of these drugs contributes to carcinogenesis in vivo, however, remains unstudied.

A primary goal of this high impact/high-risk project, a collaboration across cancer and neuroscience research groups, is to validate a new in vivo model for HNSCC.   We will utilize a well-established self-administration paradigm to deliver nicotine, or ethanol (EtOH), or both compounds in drinking water to mice; under these controlled exposures, mice will be treated with a chemical carcinogen which is known to induce the development of oral cavity squamous cell carcinoma.  This high impact/high-risk project will test the overall hypothesis that nicotine and EtOH synergistically accelerate the development of 4NQO-induced HNSCC in vivo.   Additional aspects of this high-impact/high-risk project are to determine which subtypes of nAChRs are mechanistically important for HNSCC development and progression in this in vivo rodent model.

 

Selected Publications

Dineley-Miller K.T., and Patrick J. Gene transcripts for the nicotinic acetylcholine receptor subunit beta4, are distributed in multiple areas of the rat central nervous system.  Mol. Brain Res. 16:339-344; 1992.

Segulela P., Wadiche J., Dineley K.T., Dani J., and Patrick J. Molecular cloning, functional properties, and distribution of rat brain alpha7: a nicotinic cation channel highly permeable to calcium.  J. Neurosci. 13:596-604; 1993.

Neff S., Dineley K.T., Char D., Quik M., and Patrick J. Production of polyclonal antisera that recognize and distinguish between the extracellular domains of neuronal nicotinic acetylcholine receptor subunits.  J. Neurochem. 64:332-339; 1995.

Dineley K.T. and Patrick J.W., Amino acid determinants of alpha7 nicotinic acetylcholine receptor surface expression. J. Biol. Chem. 275:13974-13985; 2000.

Dineley K.T., Westerman M., Bui D., Bell K., Hsiao Ashe K. and Sweatt J.D.  Beta-amyloid activates the mitogen-activated protein kinase cascade through hippocampal α7 nicotinic acetylcholine receptors: in vitro and in vivo mechanisms related to Alzheimer’s disease. J. Neurosci. 21:4125-4133; 2001.

Dineley K.T., Xia X., Bui D., Sweatt J.D. and Zheng H.  Accelerated plaque accumulation, associative learning deficits and up-regulation of α7 nicotinic acetylcholine receptor protein in transgenic mice co-expressing mutant human presenilin 1 and amyloid precursor proteins. J. Biol. Chem. 277:22768-22780; 2002.

Dineley K.T., Bell K.A., Bui D., and Sweatt J.D. β-amyloid peptide activates α7 nicotinic acetylcholine receptors expressed in Xenopus oocytes.  J. Biol. Chem. 277:25056-25061; 2002.

Bell K.A., O’Riordan K.J., Sweatt J.D., and Dineley K.T. MAPK recruitment by β-amyloid in organotypic hippocampal slice cultures depends on physical state and exposure time. J. Neurochem. 91:349-361; 2004.

Dineley, K.T., Hogan, D., Ru, W., Taglialatela, G. Acute inhibition of calcineurin restores associative learning and memory in Tg2576 APP transgenic mice.  Neurobiol. Learn. Mem.  88(2):217-24, 2007. PMCID: PMC2031869

Taglialatela, G., Hogan, D., Ru, W., Dineley, K.T. Intermediate- and long-term recognition memory deficits in Tg2576 mice are reversed with acute calcineurin inhibition. Behav. Brain. Res.  200(1):95-9, 2009. PMCID: PMC2663011

Hernandez, C.M., Kayed, R., Zheng, H., Sweatt, J.D., Dineley, K.T.  Loss of α7 nicotinic receptors enhances Aβ oligomer accumulation, exacerbating early-stage coginitive decline in a mouse model for Alzheimer’s disease. J. Neurosci. 30(7):2442–2453, 2010. PMCID: PMC2947456

Dineley, K.T., Kayed R., Neugebauer V., Fu Y., Zhang W., Reese L.C., Taglialatela G. (2010) Amyloid-beta Oligomers Impair Fear Conditioned Memory in a Calcineurin-Dependent Fashion in Mice.  J. Neurosci. Res. 88(13):2923-32, 2010. PMCID: PMC2919647

Rodriguez-Rivera, J., Denner, L., Dineley, K.T. (2011) Rosiglitazone Reversal of Tg2576 Cognitive Deficits is Independent of Peripheral Gluco-Regulatory Status. Behav Brain Res. 216(1):255-61, 2011. PMCID: PMC2975872

Parri H.R., Hernandez C.M., Dineley K.T. (2011) Research update: Alpha7 nicotinic acetylcholine receptor mechanisms in Alzheimer's disease.  Biochem Pharmacol. 82(8):931-42. PMID: 21763291, PMCID in progress. 

 

 

E-Mail: ktdinele@utmb.edu