Endocannabinoid Signaling in the Prefrontal Cortex in Schizophrenia

DAVID VOLK, MD, PHDTRANSLATIONAL NEUROSCIENCE PROGRAM

UNIVERSITY OF PITTSBURGH

Exploring the Relationship Between Cannabis Use, Cognitive Deficits and

Schizophrenia: Role of the Endogenous Cannabinoid System

Cannabis Use and Schizophrenia

Associated with worse clinical outcome

Kavanagh Drugs 2002

Prefrontal cortex-related cognitive impairments

Solowoji JAMA 2002

Exacerbates cognitive impairments in

schizophrenia

D’Souza BiolPsych 2005

Endocannabinoid (eCB) System

CB1 receptor is located on inhibitory axon terminals

Activation of the CB1 receptor suppresses the release of GABA

Endocannabinoid (eCB) System and Schizophrenia

GABA signaling is already impaired in the PFC in schizophrenia which contributes to cognitive deficits in schizophrenia

Cannabis use may worsen cognitive deficits in schizophrenia by aggravating pre-existing deficits in GABA signaling

Alterations in eCB signaling may worsen impairments in GABA signaling in schizophrenia

What is the status of eCB signaling in schizophrenia?

Lower CB1 receptor mRNA and protein levels in the PFC in schizophrenia (Eggan ArchGenPsych 2008)

Knowing the status of eCB signaling requires knowledge of the regulation of the eCB ligands that bind to the CB1 receptor

2-AG: The Brain’s Own Cannabis

2-AG (2-arachidonyloglycerol) is an endogenous cannabinoid

2-AG is synthesized by diacylglycerol lipase (DAGL)

2-AG is metabolized by monoglyceride lipase (MGL)

2-AG activates CB1 receptors and reduces the release of GABA

Endocannabinoid Signaling Suppresses GABA Release

Subject Demographics

Control Schizophrenia T Value(df=82)

P Value

N 42 42

Sex 31M/11F 31M/11F

Race 34W/8B 29W/13B

Age (years) 48 ± 13 47 ± 13 0.4 p=0.69

PMI (hours) 17.8 ± 5.9

18.1 ± 8.7 .17 p=0.86

Storage (mo)

97 ± 43 97 ± 46 .02 p=0.98

pH 6.7 ± 0.2 6.6 ± 0.4 1.67 p=.10

RIN 8.3 ± 0.6 8.2 ± 0.7 1.2 p=.23

Project Design

Real-time reverse transcription PCR (quantitative PCR)

RNA isolated from PFC area 9RNA is reversed transcribed to cDNAQuantitative PCR performed with SYBR Green and

gene-specific primersGene expression levels normalized to 3 reference

genes

2-AG Synthesizing and Metabolizing Enzymes

DAGLα DAGLβ MGL

ANCOVA F Stat

P Value

F Stat P Value F Stat P Value

F1,38 .019 .89 .018 .90 .841 .37

Relative Expression Level in Control Subjects

Rela

tive

Exp

ress

ion

Leve

l in

Sch

iz

Metabolizing Enzyme for Anandamide FAAH

ANCOVA F Stat

P Value

F1,38 1.1 .30

Rela

tive

Exp

ress

ion

Leve

l in

Sch

iz

Relative Expression Level in Control Subjects

Group I Metabotropic Glutamate Receptors

mGluR1α mGluR5

ANCOVA F Stat P Value F Stat P Value

F1,38 9.2 .004 3.5 .07

+12.4% +4.3%

Rela

tive

Exp

ress

ion

Leve

l in

Sch

iz

Relative Expression Level in Control Subjects

Summary of Findings

No change in 2-AG or anandamide synthesizing or metabolizing enzyme mRNA expression levels in PFC area 9 in schizophrenia. However, we do not know if changes may be still be

present at the protein level (i.e. amount and/or activity of enzyme).

Elevated mGluR1α mRNA expression levels in the PFC in schizophrenia.

Validation of Results with Additional Primers

Primer Set % Change in Schiz

Unpaired (F1,16) Paired (F1,8)

F P F P

mGluR1α_1 +38.8% 38.5 .000 37.6 .000

mGluR1α_2 +39.9% 39.1 .000 91.3 .000

mGluR1α_3 +30.7% 26.8 .000 57.2 .000

Interpreting Elevated mGluR1α mRNA Expression in Schizophrenia

Elevated signaling through mGluR1α receptors may increase 2-AG signaling, resulting in a further deleterious reduction in GABA neurotransmission in the PFC in schizophrenia.

Are elevated mGluR1α mRNA levels associated with enhanced signaling through the receptor? Elevated mGluR1α protein levels (and no change in mGluR5)

previously reported in the PFC in schizophrenia (Gupta Synapse 2005).

RGS4 and mGluR1α in Schizophrenia

RGS4 (Regulator of G Protein Signaling) reduces the duration of signaling from some G protein coupled receptors, including mGluR1α (Saugstad JNeurosci 1998).

Lower RGS4 mRNA levels have been reported in the PFC in schizophrenia (Mirnics MolPsych 2001, Erdely Synapse 2006).

Lower RGS4 and higher mGluR1α levels may together further enhance signaling through mGluR1α receptors in the PFC.

RGS4 mRNA Expression in Schizophrenia

ANCOVA F Stat

P Value

F1,38 29.4 .000

-16.1%R

ela

tive

Exp

ress

ion

Leve

l in

Sch

izop

hre

nia

Relative Expression Level in Control Subjects

*8 pairs previously studied

Interpreting Elevated mGluR1α mRNA Expression in Schizophrenia

Are elevated mGluR1α mRNA levels associated with enhanced signaling through the receptor? Elevated mGluR1α protein levels (and no change in mGluR5)

previously reported in the PFC in schizophrenia (Gupta Synapse 2005).

The combination of lower RGS4 and higher mGluR1α levels may together further enhance signaling through mGluR1α receptors in the PFC.

Novel Treatment Approaches for Schizophrenia

mGluR1α antagonist may reduce eCB-mediated suppression of GABA signaling mGluR1α /5 activation induces LTP/LTD of AMPA and NMDA

receptors (Ireland and Abraham JNeuroPhys 2009)

DAGL inhibitor would reduce suppression of GABA releaseSpecific for 2-AG and not other eCB ligands

Acknowledgements

David Lewis, MD, Director of the Translational Neuroscience Program

Translational Neuroscience Program Faculty and Staff

Liz Sengupta

Questions and Comments?

Future Studies

Cannabis use during adolescence is a risk factor for schizophrenia

Moore Lancet 2007

Mechanism of increased sensitivity to the effects of cannabis during adolescence is not understood

Knowledge of the development of the eCB system in the PFC may provide insights into mechanisms that lead to a period of increased sensitivity to cannabis