Elucidating Molecular Circuits Supporting Neuronal Signaling and Health
Blaze Light Sheet Ultramicroscope
Optical sectioning of a whole mouse brain via the Blaze Light Sheet Ultramicroscope demonstrates c-fos labeling at increasing magnification moving from A to D where individual activated cells are readily detected.
Neuronal Activation Inside a Whole Mouse Brain: c-fos labelling showing neuronal activation on a whole mouse brain. After immunolabelling and tissue clearing, brain was scanned on the Blaze Light Sheet Ultramicroscope. Credits: Lorena Areal, Paula Kurdziel and Jana Strickler.
Undergraduates & Post-bacs
Research in the Blakely laboratory focuses on the structure, regulation and disease relevance of neurotransmitter transporters, proteins that govern the inactivation of chemical signals released at synapses to elicit target activation and inhibition and ultimately control the way we think, move and behave. The work utilizes a range of physiological, optical, genetic and genomic methods to understand how changes in transporter function can support neuroplasticity as well as neural disorders. The researchers use multiple model systems that include the powerful genetic model C. elegans, where discovery of molecules that support neural signaling and health is enhanced by rapid growth and ease of genetic manipulation. Other studies utilize genetically modified mice whose complex circuitry and behaviors align with those present in humans and can allow for modeling of neurobehavioral and neurodegenerative disorders. In collaboration, the researchers pursue studies in humans exhibiting the molecular and circuit changes they have observed in animal models and aid in the development of novel therapeutics. Together, these tools, models and paradigms are pursued to advance understanding, diagnosis and treatment of brain diseases that impact the lives of millions worldwide, disorders ranging from ADHD and autism to neuropsychiatric disorders and neurodegenerative diseases such as Alzheimer's and Parkinson's disease.
Structure
Structure-Guided Drug Discovery
Synaptic transporters are vital for maintaining the ability of nerve cells and glia to use chemical messengers to communicate with one another. Over the past 30 years, my lab has focused on various aspects of transporter structure and protein interactions that permit precise localization and function of transporter proteins and their functional regulation. We know now that transporters are shuttled to synaptic areas and membrane microdomains via structural motifs evident in cytosolic sequences that can be post-translationally modified to achieve transporter regulation.听
We have identified multiple human mutations in these domains for various transporters and study the impact these mutations have on transporter structure, function and regulation to gain a glimpse into mechanisms that can support disease risk. We also gain insight into regulatory control of transporters via natural genetic variation in them across evolution.
Such variation provides a rich opportunity to understand molecular networks that control and respond to subtle and not-so-subtle changes in transporter structure. With new AI-facilitated structural folding and imaging approaches, we are pursuing opportunities for functional elements in transporters and their regulators not evident through conservation-based approaches. These models, tested for functional relevance, provide efficient templates for drug discovery that can identify therapeutic leads with greater efficiency and specificity.
Networks
Mapping Brain Networks
The Blakely lab continues to implement new approaches and technologies to elucidate molecular, cellular and circuit-level networks that support neuronal signaling and health. Using the powerful genetic model system
Caenorhabditis elegans,
and taking advantage of behaviors that emerge with loss of the dopamine transporter, we utilized unbiased genetic screens to identify and characterize genes required for the precise regulation of dopamine signaling. The genes identified in these screens have mammalian counterparts, so their identification opens up new opportunities to understand, for example, the molecular control of dopamine signaling as well as potential insights into risk for brain disorders, including Parkinson's disease, addiction, ADHD and autism.听
Other approaches take a top-down approach, where the complex fabric of neural circuitries is mined for the secrets that emerge when brain networks are probed in freely behaving animals. In mouse models, we utilize genetically encoded sensors for neurotransmitters and cell excitability to evaluate how fundamental behaviors derive from different neurotransmitters, to understand the impact of human disease-associated mutations, and to evaluate potential therapeutic leads
in vivo.
听
With whole-brain mapping of neuronal excitation and circuit responses to drugs and behavioral challenges, we are gaining insights into the circuit-level plasticities that afford resilience to stress during early life and how to capitalize on a broader perspective to monitor and treat the diseases of aging. Lastly, anatomically resolved single-cell transcriptome sequencing approaches are allowing us to broaden the impact of genes, proteins and drugs that ultimately govern behavior. These efforts produce high-content data streams that can be mined using AI-based approaches to more efficiently work our way through the myriad of next-generation, personalized treatments of brain disorders.
Enduring Impact
Transporters and Brain Development
We now know that neurotransmitters that we have studied for decades as signaling molecules at adult synapses also play important roles in brain development. To gain traction with this question, we are examining the role that synaptic transporters play in sculpting structural and functional aspects of brain development using both genetically modified mice as well as behavioral manipulations in juvenile animals.
For example, we ask whether mutations in transporter genes that impact risk for lifelong mental illnesses produce their effects in the embryo, in juvenile animals or in adults, focusing on biochemical, cellular, physiological and behavioral changes.听
Key disorders of interest are those linked to perturbations in dopamine, serotonin, acetylcholine and glutamate signaling (e.g. ADHD, addiction, autism, schizophrenia, and dystonia). Secondly, we seek to understand how early-life stress leads to long-term changes in behavior that mimics that seen in humans with depression and anxiety disorders and whether these changes reflect alterations in neurotransmitter transporter function and regulation. Finally, we seek to combine our observations from the two areas noted above to understand the intersection of genes and environment in determining disease risk.
Therapeutics
Translating Discovery into Treatments
Our understanding of synaptic transporter structure, function and regulation affords us with the opportunity to translate this knowledge to the arena of drug discovery, where leads for novel therapeutics can be developed and tested in vitro and in vivo. For example, we have developed a novel assay that permits a high-throughput screening approach to identify modifiers of acetylcholine signaling, targeting the presynaptic choline transporter (CHT).听
We have used this assay to identify multiple classes of novel molecules that appear to regulate the function of CHT and may provide new opportunities to offset changes in cholinergic signaling that arise in dystonia, Parkinson鈥檚 disease, neuromuscular, and autonomic disorders. Acting in collaboration with biotechnology and drug development companies, we are also providing critical expertise and efforts in their drug discovery projects. As we proceed further in our understanding of the contribution made by novel transporter regulators, we expect to embark on new efforts, both in house and with industry collaborators, to develop leads for novel medications.
Animal Models
Modeling Brain Disorders
Using transgenic technologies, we pursue the study of how heritable and de novo genetic variation in transporter genes leads to risk for neurological disorders. These studies initiate from identification of transporter genetic variation in humans with a range of disorders. Over the past decades we have identified such variation in subjects with autonomic dysfunction, neuromuscular disorders, ADHD and bipolar disorder, and autism. In subsequent efforts, we have generated the first mouse model of perturbed dopamine signaling in ADHD.听
Ongoing studies look to contribute both human and mouse model efforts to disorders with deficits in acetylcholine and glutamate signaling. To these studies, we bring our substantial know-how in synaptic biochemistry and anatomy as well as physiological and behavioral methods. Our goal is not to reproduce all of the facets of a given disorder in the model. Such an effort naively assumes that all facets of human physiology and behavior can be reproduced in a rodent model. Rather, we seek to understand how changes at a molecular, cellular and organismal level can emerge when synaptic transporters are not working well, leading to both new fundamental insights into potential gene networks and affording the development of novel medications that can manipulate these networks to benefit those afflicted with brain disorders. We also capitalize on a rich battery of transgenic mice (and worms) that carry specific alterations in brain signaling pathways allowing us to further dissect components of these networks, and to determine which components, where in the brain, and when in development, are effects levied to to perturb physiology and behavior.
Plasmids
DNA Constructs
The following popular plasmids created and published by the are available from
hSERT pcDNA3
plasmid #
insert: hSERT, SLC6A4, human
hNET pcDNA3
plasmid #
insert: hNET, human
mCHT pcDNA3
plasmid #
insert: hemicholinium-3-sensitive choline transporter, mouse
hChT pcDNA3
plasmid #
insert: hemicholinium-3-sensitive choline transporter, human
DAT-1 cDNA
plasmid # (available soon)
insert: dopamine transporter, from C. elegans in pCDNA3
P dat-1 ::GFP
plasmid # (available soon)
insert: dopamine transporter (DAT-1) reporter GFP fusion, C. elegans
P dat-1 ::DAT-1:GFP
plasmid # (available soon)
insert: dopamine promoter and transporter (DAT-1) translational GFP fusion, C. elegans
Antibodies
Monoclonal anti-human NET
Host: mouse
Specificity: human and non-human primates
Source: peptide, amino acids 17-33 of human norepinephrine transporter
Available at MAb Technologies: , 100 ul hybridoma media in glycerol
Monoclonal anti-rat/mouse NET
Host: mouse
Specificity: mouse and rat
Source: peptide, amino acids 05-17 of mouse norepinephrine transporter
Available at MAb Technologies:
-
, 50 ul ascites
-
, 100 ul hybridoma in glycerol
Monoclonal anti-human SERT
Host: mouse
Specificity: human and non-human primate
Source: peptide, amino acids 51-66 of human serotonin transporter
Available at MAb Technologies:
-
, 50 ul ascites
-
, 100 ul hybridoma media in glycerol
DAT-1 cDNA
plasmid # (available soon)
insert: dopamine transporter, from C. elegans in pCDNA3
P dat-1 ::GFP
plasmid # (available soon)
insert: dopamine transporter (DAT-1) reporter GFP fusion, C. elegans
P dat-1 ::DAT-1:GFP
plasmid # (available soon)
insert: dopamine promoter and transporter (DAT-1) translational GFP fusion, C. elegans
Mouse Lines
The Blakely Lab has created the following mouse lines. References detailing the generation of each of the lines are included below.
For inquiries, please contact Dr. Blakely directly.
SERT Ala56
- Gly56Ala variant is associated with autism
- Veenstra-VanderWeele J, Jessen TN, Thompson BJ, Carter M, Prasad HC, Steiner JA, Sutcliffe JS, Blakely RD. Modeling rare gene variation to gain insight into the oldest biomarker in autism: construction of the serotonin transporter Gly56Ala knock-in mouse. J Neurodev Disord. 2009 June; 1(2):158-71, [PMID: , PMCID: ]
SERT I172M
- I172M variant disrupts high-affinity binding of antidepressants and cocaine
- Thompson BJ, Jessen T, Henry LK, Field JR, Gamble KL, Gresch PJ, Carneiro AM, Horton RE, Chisnell PJ, Belova Y, McMahon DG, Daws LC, Blakely RD. Transgenic elimination of high-affinity antidepressant and cocaine sensitivity in the presynaptic serotonin transporter. Proc Natl Acad Sci USA. 2011 Mar1;108(9):3785-90, [PMID: ; PMCID:]
DAT Val559
- DAT A559V variant is associated with bipolar disorder and ADHD
- Mergy MA, Gowrishankar R, Davis GL, Jessen TN, Wright J, Stanwood GD, Hahn MK, Blakely RD. Genetic targeting of the amphetamine and methylphenidate-sensitive dopamine transporter: On the path to an animal model of attention-deficit hyperactivity disorder. Neurochem Int. 2013 Dec 8, ePub ahead of print, [PMID: ]
Floxed IL-1R
- Conditional elimination of interleukin-1 receptor
- Available from
- Robson MJ, Zhu CB, Quinlan MA, Botschner DA, Baganz NL, Lindler KM, Thome JG, Hewlett WA, Blakely RD. Generation and Characterization of Mice Expressing a Conditional Allele of the Interleukin-1 Receptor Type 1, PLoS One. 2016 Mar 1;11(3):e0150068. doi: 10.1371/journal.pone.0150068.
CHT KO
- Knockout of the hemicholinium-3 sensitive choline transporter
- Homozygous deletion of CHT is lethal
- Available from
- Bazalakova MH, Wright J, Schneble EJ, McDonald MP, Heilman CJ, Levey AI, Blakely RD. Deficits in acetylcholine homeostasis, receptors and behaviors in choline transporter heterozygous mice. Genes Brain Behav. 2007 Jul;6(5):411-24 [PMID: ]
BAC-CHT
- Multiple copies of choline transporter gene
- Available from
- Holmstrand EC, Lund D, Cherian AK, Wright J, Martin RF, Ennis EA, Stanwood GD, Sarter M, Blakely RD. Transgenic over expression of the presynaptic choline transporter elevates acetylcholine levels and augments motor endurance.Neurochem Int. 2013 Nov 22, ePub ahead of print, [PMID: ]
CHT Motorneuron Overexpressing Mice
- CHT +/+ and CHT -/- expressed under control of the motor neuron promoter Hb9
- Lund D, Ruggiero AM, Ferguson SM, Wright J, English BA, Reisz PA, Whitaker SM, Peltier AC, Blakely RD. Motor neuron-specific over expression of the presynaptic choline transporter: impact on motor endurance and evoked muscle activity. Neuroscience. 2010 Dec 29;171(4):1041-53, [PMID: ; PMCID: ]
ProT KO
- Proline Transporter knock-out mouse
- More information available soon
All of Us
2024
From left to right: Gabriella Smith (front), Erin Bell, Sofia Wasilewski, Ahmarne Hammett, Paula Gajewski-Kurdziel (front), Hideki Iwamoto, Mason Martin, Carina Meinke, Randy Blakely, Zaka Asif, Matt Schrier, Manuela Zapata Garcia, Maureen Hahn, Allison Walsh, Anna Stauffer, Siddhi Gawker (front). Not shown: Cara Melillo, Tristan Wells, Peter Rodriguez, Cristal Auger.
2016
From left to right, front row: Maureen Hahn, Catherine Nettesheim, Cassie Retzlaff, Chelsea Snarrenberg, Keeley Spiess and Nicole Baganz.
Middle row: Paul Gresh, Meagan Quinlan, Joseph Balbona, Randy Blakely and Gwynne Davis.
Last row: Peter Rodriguez, Osama Refai, Matt Gross, Hideki Iwamoto, Raajaram Gowrishankar, Adele Stewart, Matthew Robson and Isabel Stillman.
Research Staff
Anna Stauffer - Lab Manager
Anna Stauffer, a Pennsylvania native, joined the Blakely Lab in the summer of 2022. As lab manager, her goal is to ensure a safe, efficient and productive laboratory environment and to contribute to cutting-edge science. She provides orientation, training and lab administrative support for all lab members and serves as the liaison with the veterinary staff in the Division of Comparative Medicine for mouse colony management, cryopreservation and shipments. In addition to her administrative duties, she provides technical support to colleagues who use mouse models to investigate the neurological and behavioral consequences of altered neurotransmitter transporter expression and function.
Angelina Emerson - Research Assistant
Angelina earned her bachelor's degree in psychology from 麻豆精品视频 Harriet L. Wilkes Honors College, where her studies sparked a strong interest in neuroscience. During her undergraduate training, she completed an honors thesis titled
From Sleep to Synucleinopathies: REM Sleep Behavior Disorder as an Indicator of Neurodegeneration
, which examined REM Sleep Behavior Disorder (RBD) as an early clinical marker of neurodegenerative synucleinopathies such as Dementia with Lewy Bodies and Parkinson鈥檚 disease. Her work explored the evolving understanding of disease progression, the importance of early detection, diagnostic differentiation, and opportunities for earlier therapeutic intervention. In the Blakely Lab, Angelina鈥檚 research centers on serotonin signaling and neurotransmission, with a focus on how serotonin transporter dysfunction and neuroimmune interactions may contribute to the development of neuropsychiatric disorders, including depression and anxiety.
Undegraduates and Post-Bacs
Cristal Auger, undergraduate, Harriet L. Wilkes Honors College
Cristal Auger is an undergraduate researcher at the Harriet L. Wilkes Honors College, concentrating in biology and data science with a minor in business. Within the Blakely Lab, she studies dopamine (DA) signaling using the model organism
Caenorhabditis elegans
. DA signaling is integral in cognitive function, with alterations leading to brain disorders including Parkinson's disease, schizophrenia, autism, and addiction. The DA-dependent phenotype swimming-induced paralysis (Swip) indicates altered DA-signaling in
C. elegans
. DA-dependent Swip was screened for across 300 strains of the Million Mutation Project (MMP) library, a resource of chemically mutagenized and fully sequenced
C. elegans
lines. Her research in the lab consists of bioinformatic evaluations, AI analysis, and experimental trials to identify novel gene candidates responsible for altered DA-signaling in MMP lines where DA-dependent Swip has been found.
Ashley Castresana, undergraduate, Harriet L. Wilkes Honors College
Ashley is an undergraduate researcher in the Blakely Lab studying how regulation of the serotonin transporter (SERT) shapes social behavior. SERT plays a critical role in maintaining serotonin homeostasis, and alterations in serotonin signaling have been implicated in multiple neuropsychiatric disorders characterized by changes in social function. Ashley contributes to studies of a genetically modified mouse model carrying an alanine substitution at SERT threonine 276 (SERT T276A), a site required for protein kinase G (PKG)-dependent regulation of SERT. Studies of these mice have revealed sex-dependent behavioral alterations, including reduced sociability in females. Building on these findings, Ashley鈥檚 project investigates whether disruption of SERT Thr276 regulation also alters maternal care, allowing her to determine whether the female social phenotype extends beyond sociability to social nurturing behaviors. Through behavioral studies of T276A females and their offspring, her work seeks to better understand how altered regulation of serotonin signaling influences distinct dimensions of female social behavior.
Ahmarne Hammett, undergraduate, Harriet L. Wilkes Honors College
Ahmarne Hammett is an undergraduate student at the Harriet L. Wilkes Honors College, majoring in cellular neuroscience with a minor in interdisciplinary ethics. Within the Blakely Lab, she assists with worm preparation, whether NGM agar plates or simply seeding these plates with
E.coli
bacteria. Alongside this, she assists with experiments and follow through with any protocols needed by her supervisor, Dr. Zaka Asif, such as performing bleach synchronization or preparing L4 worms for microscopic studies.
Samuel Mittleman, undergraduate, Harriet L. Wilkes Honors College
Sam Mittleman is an undergraduate student at the Harriet L. Wilkes Honors College, majoring in cellular neuroscience. Within the lab he is working to understand the connection between inflammation, the serotonin system, and neuropsychiatric disorders with a special interest in autism spectrum disorder with Dr. Paula Kurdziel. He helps with performing tasks and experiments in the lab. In his free time he enjoys reading books, playing games, and going out with friends.
Randy D. Blakely, Ph.D.
Executive Director
麻豆精品视频Stiles-Nicholson Brain Institute
David J.S. Nicholson Distinguished Professor in Neuroscience,
Professor, Dept Biomedical Science
Charles E. Schmidt College of Medicine
