preclinical models for the study of rhythms and · methods of manipulation 1. gene targeting,...
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Preclinical models for the study of circadian rhythms and
diseaseColleen A. McClung, Ph.D.
Professor of Psychiatry and Clinical and Translational ScienceUniversity of Pittsburgh School of Medicine
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The health consequences of shortened or reduced sleep and desynchronized circadian rhythms
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From: The Pathophysiologic Role of Disrupted Circadian and Neuroendocrine Rhythms in Breast CarcinogenesisEndocr Rev. 2016;37(5):450-466. doi:10.1210/er.2015-1133Endocr Rev | Copyright © 2016 by the Endocrine Society
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SCN afferent and efferent pathways
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A Distributed Network of Circadian Clocks
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The Period Gene‐‐‐ 2017 Nobel Prize in Medicine!Summary of Early Findings
• Per is the first single gene shown to control behavior.
• PER protein is located in the nuclei of brain and visual neurons.
• Per transcription, translation and nuclear localization is rhythmic.
• Accumulation of PER suppresses its own transcription
• PER binds to other factors to control transcription
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The Clockmutant mouseClock was identified in a screen of mutagenized mice done in the labs of Fred Turek and Joe Takahashi (Vitaterna et al.,1994).
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or NPAS2
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Circadian rhythms and metabolic disorders
• Animal models are used in multiple ways to understand the association between circadian rhythms and obesity, diabetes, and other metabolic disorders
• Circadian gene mutation/knock out/knock‐in‐‐‐‐ How does this influence feeding, metabolism, microbiome, glucose, weight gain etc.
• High fat, high calorie or high sugar diet‐‐‐‐ Does this change circadian rhythms or the molecular clock, resulting in metabolic phenotypes?
• Restricted feeding‐‐‐‐ Does eating at a particular time of day lead to better or worse metabolic outcomes? How does this relate to normal rhythms in the liver, fat cells, etc.?
• Environmental rhythm disruptions‐‐‐‐ Does a jet lag paradigm, light at night, or other rhythm disruption lead to altered metabolic phenotypes?
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Methods of manipulation
1. Gene targeting, transgenics2. Pharmacological manipulation3. Antisense oligonucleotide, antibodies4. Viral gene transfer5. RNAi 6. Optogenetics7. DREADDs
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Circadian Rhythms and Cancer or immune function• Animal models are used in multiple ways to understand the association between circadian rhythms and cancer and immune function
• Circadian gene mutation/knock out/knock‐in‐‐‐‐ How does this influence tumor growth or immune function?
• Environmental rhythm disruptions‐‐‐‐ Does a jet lag paradigm, light at night, or other rhythm disruption lead to altered tumor growth or immune phenotypes?
• Effect of abnormal cell growth on the molecular clock‐‐‐‐ Once a cell starts to inappropriately divide, does it lose the molecular clock, leading to further growth?
• Does the immune system and/or cell cycle have a normal rhythm?‐‐‐‐ Are animals more or less susceptible to infection or drugs targeting the cell cycle (i.e. chemotherapy) at particular times of day?
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Circadian genes and the cell cycle
Fu and Kettner, 2013
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LOSS of circadian rhythm in cytotoxicity of nkcells after jet lag
*, different from lowest value in CONT; a, different from lowest value in SHIFTED; #, between CONT and SHIFTED groups at CT Logan et al., 2012
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CONTROL CONTROL SHIFTED
SUPPRESSED NK CYTOLYTIC ACTIVITY LEADS TO INCREASED TUMOR GROWTH IN ANIMALS EXPOSED TO JET LAG
Logan et al., 2012
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Circadian Rhythms and Neurological Disorders• Animal models are used in multiple ways to understand the association between circadian rhythms and neurological disorders like Alzheimer’s and Parkinson’s Disease
• Circadian gene mutation/knock out/knock‐in‐‐‐‐ How does this influence Neuronal stress, activity, motor abilities and neuronal death?
• Environmental rhythm disruptions‐‐‐‐ Does a jet lag paradigm, light at night, or other rhythm disruption lead to increased susceptibility to neuronal death?
• Effect of age on the molecular clock‐‐‐‐Most neurological disorders are associated with advanced age. Do age related changes to the clock lead to vulnerability for neuronal death?
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Rhythm disruption increases the progression of neuronal loss
Musiek, 2017
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Loss of BMAL1 or CLOCK/NPAS2 leads to massive increases in oxidative stress and neurodegeneration
• Circadian clock proteins regulate neuronal redox homeostasis and neurodegeneration.
• Musiek ES, Lim MM, Yang G, Bauer AQ, Qi L, Lee Y, Roh JH, Ortiz‐Gonzalez X, Dearborn JT, Culver JP, Herzog ED, Hogenesch JB, Wozniak DF, Dikranian K, Giasson BI, Weaver DR, Holtzman DM, Fitzgerald GA.
• J Clin Invest. 2013 Dec;123(12):5389‐400. doi: 10.1172/JCI70317. Epub 2013 Nov 25.
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Circadian rhythms and Psychiatric DisordersAnimal models are used in multiple ways to understand the association between circadian rhythms and psychiatric disorders such as autism, depression, bipolar disorder, schizophrenia, substance use disorders
• Circadian gene mutation/knock out/knock‐in‐‐‐‐ How does this influence Neuronal stress, neuronal activity, mood, cognitive, anxiety‐like, reward related behavior?
• Environmental rhythm disruptions‐‐‐‐ Does a jet lag paradigm, light at night, or other rhythm disruption lead to altered behaviors and neuronal activity, neurotransmission?
• Molecular clock and rhythm changes across development‐‐‐Most psychiatric disorders have a developmental component, either early on or during adolescence. Rhythm changes during these critical periods could lead to vulnerability for psychiatric disorders.
• Impact of chronic stress, alcohol or drugs of abuse on rhythms‐‐‐‐ These factors can disrupt normal rhythmicity in the brain and in circulating hormones that influence mood, motivation, reward value, etc.
• Impact of altered diurnal rhythms in neuronal activity‐‐‐‐ Techniques like optogeneticsand DREADDs can be used to directly influence normal rhythms of neuronal activity in multiple brain regions to determine how this influences behavior and other features of psychiatric disorders.
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Clock mutant miceHyperactivity
Sleep less than wild type mice
Less depression-like behavior in a variety of models
Increased novelty seeking, impulsivity and risk taking
Are more sensitive to the rewarding effects of cocaine, sucrose, and brain
stimulation
Bipolar patientsHyperactivity
Decreased need for sleep
Feelings of euphoria
Excessive involvement in activities that have a high potential for painful
consequences.
Propensity towards drug use and abuse
The Clock mutant mice are very similar to bipolar patients in the manic state
Roybal et al., Proc Natl Acad Sci (2007)
Lithium or VPA treatment reverses these phenotypes
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Clock∆19 mice exhibit increased ethanol intake
Ozburn et al., Neuropsychopharm, 2013
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Clock knock‐down in the VTA increases alcohol preference
Ozburn et al., Neuropsychopharm, 2013
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BRAIN REWARD REGIONS
Nestler et al., (2003)
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Dopamine is important in psychiatric disorders• Mania is associated with increased dopaminergic transmission in striatal regions, while some models of depression produce decreased dopamine.
• Antipsychotic drugs antagonize Drd2 receptors
• All drugs of abuse activate the VTA dopamine system. Stimulants like cocaine directly bind to the dopamine transporter
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Clock mutant mice have an increase in VTA dopamine cell firing and thisIs rescued by chronic lithium treatment
Coque et al., Neuropsychopharm (2011)
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Clock knockdown mice have higher rates of dopamine cell firing
Mukherjee et al, Biological Psychiatry, 2010
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Viral expression of functional CLOCK in the VTA is able to rescue their behavioral abnormalities
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Roybal et al., Proc. Natl. Acad. Sci. (2007)
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Clock19 mice have a large increase in daytime dopaminergic activity
B
Sidor et al., Mol Psych 2015
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JOÃO PEÇA & GUOPING FENG
Can we cause a mouse to be “manic” by stimulating dopamine neurons during the light cycle?
Step Function Opsins (SFO) allow chronic stimulation paradigms with single light pulses because they cause a gradual increase in neuronal depolarization, increasing the probability of the neuron firing in response to endogenous stimuli over 20–30 minutes.
Yizhar et al. Nature 2011
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SSFO expression in dopamine neurons
DIO-SSFO-eYFP
eYFP TH
DAPI Overlay
ITR ITRpolyAWPREEf1α SSFO-eYFP
Lox2722 sites
LoxP sites
Ef1α SSFO-eYFP
Cre
SSFO=stable step function opsin
AAV5-DIO-Ef1a-SSFO-eYFP
TH::Cre
Sidor et al., Mol Psych 2015
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SurgeriesTH::Cre mice
AAV5-SSFO-eYFP/AAV5-eYFP
4 weeks 24 hrs
Elevated plus maze
48 hrs 48 hrs
Open field
“Booster” Stim
“Booster” Stim
7-day chronic stimulation
ZT 6-10 (daytime)
ZT 18-22 (nighttime)
Light/dark
No optical stimulation given on day of, or during, behavioral testing
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Daytime Stim Nighttime Stim
Locomotor response to novelty is increased with both day/night stimulations
Sidor et al., Mol Psych 2015
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Daytime Stim Nighttime Stim
Anxiolytic effects are only produced with daytime stimulation in the EPM
Sidor et al., Mol Psych 2015
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Daytime Stim Nighttime Stim
Anxiolytic effects are only produced with daytime stimulation in the D/L and OF
Sidor et al., Mol Psych 2015
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Diurnal variation in evoked firing rates of NAcMSNs
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Parekh et al., Biological Psychiatry, 2017
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Conclusions• Animal models can be used to understand the mechanistic links between circadian rhythms and a host of diseases.
• This can be done via genetic manipulations or environmental manipulations of the circadian clock
• Many models also look at the response of the clock to some sort of paradigm such as chronic stress, high fat diet, or exposure to drugs of abuse
• Tools like optogenetics and DREADDs can be used to manipulate rhythms in neuronal activity in the brain to understand how these patterns are associated with behavior and other measures.
• It’s important to look at rhythms in wild type animals to understand how processes are normally regulated over the 24 hr cycle and to look across development, aging and at sex as a biological variable.
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