Nanoparticles and Gene Therapies for Addictions: What to Do with DARPP-32

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1 Nanoparticles and Gene Therapies for Addictions: What to Do with DARPP-32 Bruce A. Maslack, MD ASAM Ruth Fox Course Date April 15, 2010

2 Financial Disclosures Reckitt- Benckiser Speaker Honoraria Forest Pharmaceuticals Speaker Honoraria

3 My Other Disclosures I am a clinician, not a Credentialed Scientist or Researcher, though I am an admirer and attentive follower of those who are I am on the Ruth Fox Course Planning Committee I opened my mouth a year ago when we were discussing a Hot Topic to present this year I couldn t deliver the presenter

4 A Dedication... To Paul Greengard, Ph.D. With Arvid Carlsson and Eric Kandel won the Nobel Prize in Physiology or Medicine in 2000 For showing how neurotransmitters act on the cell and can activate a central molecule known as DARPP-32 According to Wikipedia once a notable potato sack racer.

5 To Fight Drug Addiction, UB (University of Buffalo) Researchers Target the Brain with Nanoparticles. March 23, Bonoiu, A.C. et al. PNAS Online.

6 So what are they doing in Buffalo and why? Building a stable nanoparticle to cross the blood-brian barrier Delivering short, silencing RNA molecules to cell nuclei...turn off a gene that plays a critical role in many kinds of drug addiction And this alcohol and drug research is funded by the National Cancer Institute

7 Getting Ready to get to Buffalo...

Implanted so that semi-permeable probe tip is in specific brain region of interest.

8 Sampling of Interstitial Neurochemicals Allows sampling of neurochemicals in conscious animals (correlate brain chemistry with behavior). Implanted so that semi-permeable probe tip is in specific brain region of interest. Substances below the membrane MW cutoff diffuse across membrane based on concentration gradient. Both neurochemical sampling and localized drug delivery are possible.

Dopamine Neurotransmission frontal cortex % of Basal Release 1100 1000

After Amphetamine nucleus accumbens VTA/SN % of Basal Release 200 150

9 Dopamine Neurotransmission frontal cortex % of Basal Release AMPHETAMINE hr Time After Amphetamine nucleus accumbens VTA/SN % of Basal Release Empty Box FOOD Feeding Time (min) Di Chiara et al.

10 Acc VTA FCX AMYG VP ABN Raphé LC GLU GABA ENK OPIOID GABA GABA GABA DYN 5HT 5HT 5HT NE HIPP PAG RETIC To dorsal horn END DA GLU Opiates ICSS Amphetamine Cocaine Opiates Cannabinoids Phencyclidine Ketamine Opiates Ethanol Barbiturates Benzodiazepines Nicotine Cannabinoids OPIOID HYPOTHAL LAT-TEG BNST NE CRF OFT MesoLimbic Dopaminergic Circuit Pleasure/Reward Center

11 So what did I just say about? The Ventral Tegmental area? The Nucleus Accumbens? The Extended Amygdala? The Medial Prefrontal Cortex? The Substantia Nigra? The Ventral Striatum?

12 Neuroanatomical Structures and their Significance for Addiction Nucleus Accumbens and Central Nucleus of the Amygdala: Forebrain structures involved in the rewarding effects of drugs. Key reward neurotransmitters - dopamine and opioid peptides Extended Amygdala: Includes central nucleus of the amygdala, bed nucleus of the stria terminalis and a transition zone in the nucleus accumbens. Contains corticotropin releasing hormone (CRH) Medial Prefrontal Cortex: Substrate for Executive function. Contains major NMDA/glutamate projections to the nucleus accumbens and amygdala (Koob, 2005)

13 From this point on I d be nowhere without these papers... Heilig, Markus, et al, Translating the Neuroscience of Alcoholism into Clinical Treatments: Blocking the Buzz to Curing the Blues. Biobehavioral Review (2009) Vengeliene, V. et al, Neuropharmacology of Addiction. British Journal of Pharmacology (2008)

14 The Dopamine-Centric View of Addiction DA Pathway a critical substrate for motivation to engage in exploration and goal-oriented behavior Most, if not all drugs of abuse, interact with this DA circuitry Certain drugs can activate DA signaling in a supra-physiological amplitude; may successfully compete with natural rewards

15 There s More to Addiction than Reinforcement Acute positively-reinforcing effects of addictive drugs play a role in early stages of addiction Addiction, as a Chronic, Relapsing Disorder, is ultimately-maintained by a pathology of brain function arising over time, and only in a minority of subjects that have initiated drug use (McLellan, et al, 2000) Drug-seeking and taking become habitual, essentially disengaged from normal motivational control (Belin, et al, 2009)

16 The Dopamine Story Refined Conditioned reinforcement from drug-related cues From Reward to Expectation of Reward - Pavlovian conditioning Incentive Salience for drug-associated stimuli, even while actual drug reward undergoes tolerance Pre-existing differences in DA receptor endowment determine vulnerability to drug reinforcement

17 A Better Realization That: DA serves as a direct indicator of reward only, at-best, in early phases of addiction In the long-term, the most important role of DA is to mediate associative learning by increasing salience to environmental cues present during drug taking This marvelous success (at uncovering the physiology of brain systems that mediate natural reward and learning) has not brought us major advances in the treatment of addictive disorders (Heilig, 2009)

18 Let s Take Alcohol for an Example...

19 The Neuroscience of Alcoholism What are the Factors (Genetic or Environmental) Involved in the Initiation and Maintenance of Alcohol Consumption? What are the Changes to the Brain that Underlie the Transition from Controlled to Compulsive Use?

20 Specific Targets of Alcohol Receptors: Ionic Channels: NMDA/Glutamate L-Type Calcium Channels GABAa Glycine G-Protein-Activated Inwardly Rectifying Potassium Channels 5-HT3 Nicotinic ACh (Vengeliene, 2008)

21 How is Alcohol Rewarding? Microdialysis studies (in rodents) show that alcohol releases DA at the Nucleus Accumbens - but a lower response than with amphetamine Much greater DA release in genetically-selected EtOH-preferring rats fmri shows Ventral Striatum activation in non-dependent humans with a pharmacokinetically-controlled alcohol i.v. infusion PET displacement of C-raclopride, a DA ligand also shows modest DA release to orally-administered alcohol in social drinkers fmri measures of VS activation in alcoholics with the i.v. alcohol infusion is much less pronounced

22 [11C]Raclopride Binding In Cocaine Abusers (n=18) Viewing a Neutral and a Cocaine-Cue Video Neutral video Viewing a video of cocaine scenes decreased specific binding of [11C]raclopride presumably from DA increases Volkow et al. J Neuroscience 2006.

23 So is that it for Alcohol? Yes, alcohol appears able to activate DA release but not very potently Is alcohol-related DA release causally-related to alcohol seeking and use? If it motivates alcohol seeking, does this persist into the addicted state? It has been long-known near-complete lesions of DA input to the NA in rats leave alcohol seeking and operant responses unaffected And what about that fmri study showing little Ventral Striatum activation in alcoholics? (Gilman, et al, 2009)

24 Alcohol and the Naltrexone Story Indirect DA release via DA neuronal disinhibition by release of endogenous opioid peptide Two decades after O Malley s and Volpicelli s papers (1992) - 30 randomized trails support the efficacy of the use of naltrexone But, the effect-size is modest... Or, reflects heterogeneity of response among patients... Clinical and research experience support the latter interpretation

25 The Opiate Connection: The Research Disruption of Mu-opioid receptor gene (OPRM1) causes loss of alcohol self-administration in mice Replicable association, in Swedish cohorts with little ethnic admixture, between OPRM1 118G polymorphism and addictive disorders (Bart, et al, 2004, 2005) Secondary analyses of published trials, including Project Combine, suggest OPRM1 118G carriers are particularly responsive to naltrexone; though results aren t always consistent An OPRM1 polymorphism exists in Rhesus monkeys, and classic reward occurs in 77G carriers, suppressible with naltrexone

26 Low Innate Sedative-Ataxic Response to Alcohol Studies in offspring of alcoholics have indicated this trait as a key inheritable susceptibility factor for alcohol use disorders (Schuckitt, 2009) How does this relate to other pharmacogenetic variations in alcohol responses? Could it relate to OPMR1 118G? Separate, independent inheritable factors vs. identically derived from OPMR1 118G vs. two traits functionally linked Interact to determine that an individual can consume alcohol to a level that will provide sufficiently potent rewarding action

27 Glutamate and the LORR Loss of Righting Reflex Get a mouse drunk, wait for return of the righting reflex, Inject glutamate intra-cerebroventricularly and the mouse falls over again This is evidence that glutamate enhances the central depressant effect of alcohol

28 Well, I didn t find a mouse...

29 And what can you prove with Ketamine? Ketamine is an NMDA/Glutamate receptor antagonist Healthy individuals having a (+) FH alcoholism are less responsive to ketamine than individuals having (-) FH Mice bred to be more sensitive to alcohol are also more sensitive to ketamine treatment NMDA/Glutamate receptor antagonists can inhibit alcohol-induced conditioned place preference in rats

30 Conditioned Place Preference No rat was restrained in the making of the advertisement photo...

31 Glutamate Response Tightly- Linked to Nitric Oxide Pathway Glutamate NMDAr Calcium influx Calmodulin NO Synthetase NO NO Synthetase Knock Out mice show changes in the duration of LORR induced by alcohol and the degree of alcohol reinforcement

32 And then There s GABA... High alcohol-sensitive and low alcohol-sensitive rat lines have marked differences in GABA-mediated events GABAa receptor knock out mice show strong evidence this variability is due to the alpha-1 subunit (measured by differences in LORR) Activation of the GABAb receptor suppresses acquisition of alcohol drinking behavior in rats

33 The Rewards of Alcohol(ism) Alcohol reward relies on alcohol-induced release of endogenous opioid peptides and their action is to release mesolimbic DA This is more pronounced among drinkers early on and not so evident in later addiction stages Reward in the classic sense most prevalent and perhaps persistent in those with a certain genetic endowment Those with this genetic endowment benefit from blocking the opioid effect. Naltrexone treatment may be of limited utility to others

34 Maintaining Alcohol Use When Alcohol Actions Aren t Rewarding Something kicks in as alcoholism develops This is significant for a minority of alcohol users Long-term changes affecting brain functioning induce and maintain the addicted state

35 Modeling Neuroadaptation Mice, rats and most humans won t drink enough because it simply isn t a very potent reinforcer for them With vapor inhalation obtain prolonged brain exposure that emulates clinical alcoholism Prolonged brain exposures to intoxicating levels leads to persistent behavioral consequences

36 Two Key Behavioral Consequences Escalation - Progressive increases in the voluntary intake of alcohol Sensitization to behavioral stress responses

37 Post-Dependence? The sum of neuroadaptations as an individual becomes dependent on alcohol The extent and duration of these changes are variable - indefinite in some, remitting in others

38 Persistent Sensitization to Stress Has a dynamic nature Anxiety resolves clinically in most patients in 3-6 weeks Plus Maze findings in post-alcohol-exposed rats correlate to this, but PM after Restraint Stress gives a very different picture. This is a persistent effect What if we could select a stressor that did not effect anxiety in animals w/o alcohol exposure? In many instances these stressors have a potent anxiogenic effect in the alcohol-exposed

39 Will he? or won t he? Balancing security with the urge to explore...

40 Up-Regulated Brain Responses in Detoxed Alcoholics International Affective Picture System and fmri - Shows sensitized activation especially in the Insula Insula activation correlates to subjective measures of craving in other studies - Encodes negative interoceptive states. Sensitized brain response persists three weeks at minimum Post-alcoholics not necessarily characterized by anxiety

41 So Is it Up-Regulated Reactivity to Stress? The common view is that stress increases voluntary alcohol intake That doesn t happen to non-dependent animals The response is very different after a history of alcohol dependence Non-stressed - Greater voluntary alcohol consumption Stressed - Intake of alcohol escalates Remove stress - Maintain the higher level of drinking

42 What s Happening Here? Long-term neuroadaptations in alcohol dependence not only lead to escalation of alcohol intake, But also to sensitization of stress responses And neuroadaptation creates the link between these two phenomena

43 Alcoholism and CRH Outside the Hypothalamus Extensive networks of neurons express Corticotropin Releasing Hormone (CRH) in the Extended Amygdala - a critical area for stress responses and emotionality CRH-1. Substantially accounts for the stress response CRH-2. More variable and region-dependent. Effects generally opposite to CRH-1 receptors An Alarm System - normally quiescent - signaling only by exposure to overwhelming stress

44 When CRH is Activated (in the Extended Amygdala) Induces acute alcohol withdrawal anxiety Persistent sensitization of behavioral stress responses CRH-mediated Escalating alcohol self-administration in the WD state CRH-mediated As is alcohol self-administration long after WD in the post-dependent These are equally sensitive to CRH or CRH-1-specific antagonists No CRH antagonist has any effect on drinking in non-dependent animals

45 How does this come about? Increased CRH release in the CeA in acute WD correlates with decreased CRH tissue levels of CRH in acute withdrawal 6 weeks post-wd tissue CRH expressed in supranormal amounts There is increased CRH signaling Increased CRH transcript levels are demonstrated in the postdependent state (Heilig, et al, in press) The major contributor to CRH signaling is up-regulation of CRH-1 receptor expression and CRH-1 binding in the Amygdala

46 Marchigian-Sardinian Preferring (msp) Rats Genetically selected for alcohol preference A Phenocopy of post-dependent rats; have the same increased stress reactivity, excess self-administration and increased propensity for modeled relapse-like behavior Marked up-regulation of the transcript encoding for the CRH-1 receptor in the amygdala Crhr-1 promoter is unique to msp rats Selective CRH-1 antagonist, Antalarmin, blocks phenotypic expression

47 msp Rats and Post-Dependent Rats Given ad lib access to alcohol, consumption down-regulates the receptor transcript to normal levels in msp rats In genetically non-selected rats in the post-dependent state, the transcript up-regulation persists long after alcohol exposure This is most pronounced in the basolateral and medial amygdala Microinjections of CRH-1 antagonists are being performed to map this out

48 Pexacerfont (BMS-562,086)? CRH-1 antagonist (like Antalarmin) Bristol Myers Squibb has this substance currently in clinical trials for the treatment of anxiety disorders Also proposed for the treatment of depression and irritable bowel syndrome

49 Substance P (SP) and Neurokinin-1 (NK-1) Receptors The NK-1 Story especially is as interesting as CRH It s presented well in Heilig (2009), but not here in my lecture

50 Neuroadaptation All drugs of abuse interact initially with a protein extracellularly on the membrane But the many effects drugs of abuse exert on the brain are via perturbations of intracellular messenger pathways Old view: Neurotransmitter-receptor interaction causes +/- influences on ion channels,promoting or attenuating nerve impulses New view: Every process in a neuron is influenced by neurotransmitter-receptor signaling

51 Complex Cascades Intracellularly First Messenger: The neurotransmitter Coupling Factors: G (guanine-binding) protein - influences ion channels and secondary messenger levels Secondary Messengers: camp, Ca++, nitric oxide, prostaglandins, inositol triphosphate -effect changes in protein phosphorylation; adding or removing a phosphate group

52 Protein Phosphorylation is the Major Currency of Cells Phosphate groups are bulky and highly charged Adding/Subtracting phosphate alters a Protein s activity 1. Receptor +/- phosphorylation - Alters transmitter receptivity 2. Ion channel +/- phosphorylation - Alters conductance 3. Enzyme +/- phosphorylation - Affects neurotransmitter synthesis 4. Ribosomal +/- PO4 - Alters protein synthesis 5. Transcription Factors +/- PO4 - Alters gene expression

53 This Brings Us to DARPP-32 Dopamine, camp-regulated Phosphoprotein of 32,000 kda A key Tertiary Messenger - it s state of phosphorylation integrates much that influences DA cells through neurotransmission, other neuromodulators Activated Protein Kinases A or G - Phosphorylates at Thr34, converts DARPP-32 into a potent inhibitor of Protein Phosphatase -1 (PP-1) DARPP-32 phosphorylation at Thr75 converts it into an inhibitor of PKA (by inhibiting phosphorylation at Thr34) Unique as a dual-functioning protein

54 Where s DARPP-32? The Striatum (Caudate - Putamen and Nucleus Accumbens) Olfactory Tubercle Bed nucleus of the Stria Terminalis Portions of the Amygdala Moderate amounts of DARPP-32 throughout the Neocortex, especially layers II, III, and VI; & dentate gyrus of Hippocampus Distribution similar in all species studied; of mice and men

55 Effects of Dopamine and Glutamate on DARPP-32 DA, activating D1 receptors and adenylate cyclase through AC - stimulatory G-Protein, turns on PKA phosphorylation of DARPP-32 DA, acting through D2 receptors and AC inhibitory G-Protein reduces DARPP-32 phosphorylation Calcineurin - a principal mediator of dephosphorylation and inactivation of DARPP-32 Glutamate dephosphorylates DARPP-32, presumably through calcium-dependent activation of Calcineurin

56 Diagram of DARPP-32 Gould T D, Manji H K PNAS 2005;102: by National Academy of Sciences

57 ERK? Extracellular signal-regulated kinase A down-stream effector of DARPP-32 ERK and related kinases have documented effects on learning and memory D-amphetamine given to mice increases phosphorylated (activated) ERK. This effect is not seen in DARPP-32 knockout mice DARPP-32 knockout also reduces Nucleus Accumbens ERK activation with cocaine, nicotine, morphine, and THC

58 What Else Could ERK Proteins Do? ERK is a ubiquitous signaling pathway. Can it play only a single specific role? Different ERK-positive cells have different inputs, outputs and functions Signaling systems are anchored and clustered. Subcellular localization may provide specificity ERK cascade has well-defined modules with input, output, feedback loops, and multistep regulatory controls Learned behavior of biological systems may well be stored within intracellular biochemical reactions of signaling pathways like ERK

59 Gene Therapies Targeting DARPP-32 (We re in Buffalo Now) An interdisciplinary team (Mahajan, et al, 2009) published an initial study treating cell cultures of normal human astrocytes with heroin, assaying for modulation of cellular proteins, finding increased expression of DARPP-32 especially. Cells were treated with siport (an Ambion, Inc. product) - essentially an artificial liposome that incorporates with the cell membrane introducing sirna specific for DARPP-32 sirna-treated cells were then treated with heroin and gene expression levels of DARPP-32, PP-1, ERK, and CREB were assayed using quantitative polymerase chain reaction assay

60 sirna? Small Interfering RNA First discovered in 1993 Interfere, silence or quell the expression of messenger RNA Micro RNA s (mirna), Small RNA s (sirna), and Piwi-interacting RNA s (pirna)

61 RISC? RNA interference pathway in many eukaryotes Initiated by Dicer RNA-induced silencing complex Base pair matching, cleavage by Argonaute This won the 2006 Nobel Prize for Andrew Fire & Craig Mello

64 The Nanotechnology Approach Biocompatible Gold nanorods are given a cationic charge sirna is readily adherent forming Nanoplexes, stable a month The nanoplexes can be visualized entering cultured dopaminergic cells DARPP-32 and downstream effector molecules expression reduced to background levels, and superior to siport- mediated transfection Effective at crossing an established in vitro model of the BBB

65 What s Next? Preparing for an in vivo trial What pitfalls can you now see for this approach?

66 A plug for a new book... Advances in the Neuroscience of Addictions (Frontiers in Neuroscience). Cynthia M. Kuhn and George F. Koob (Editors). CRC Press. April 5, 2010 Encyclopedia of Behavioral Neuroscience, 3 Volumes. George F. Koob, Michel LeMoal and Richard F. Thompson (Editors). Elsevier Science. June 9, 2010

67 And What I Read Recently... Study Shows Compulsive Eating Shares Addictive Biochemical Mechanism with Cocaine, Heroin Abuse. Scripps Research Institute, March 29, What are my chances of getting this person (Paul Kenny, Ph.D.) from Florida to Washington DC for Ruth Fox 2011?

MOLECULAR BIOLOGY OF DRUG ADDICTION. Sylvane Desrivières, SGDP Centre

1 MOLECULAR BIOLOGY OF DRUG ADDICTION Sylvane Desrivières, SGDP Centre Reward 2 Humans, as well as other organisms engage in behaviours that are rewarding The pleasurable feelings provide positive reinforcement