Biology subjects

Harvey, B. K.

Publications and source records attributed to Harvey, B. K..

10 recordsLinked to original sources

bioRxiv2026

Title: Unilateral loss of tyrosine hydroxylase in adult midbrain dopaminergic neurons reveals reduction to neuregulin1 in rat model of acute dopaminergic dysfunction

Dopamine, produced primarily in the midbrain, is required for movement and reward-based signaling all throughout life. Loss of dopaminergic function can result in changes to motor systems, mood, motivation, and cognition. Dopaminergic cell death or dopaminergic hypofunction are implicated in a variety of diseases including synucleinopathies, neuropsychiatric disorders, and substance use disorders. Loss of dopaminergic neurons (DANs) in the substantia nigra pars compacta (SNpc) and subsequent dysregulation of dopamine signaling in striatum are implicated to varying degrees in all synucleinopathies. By the time patients present with motor symptoms in Parkinson's disease, the most common synucleinopathy, >30% of DANs are lost in the SNpc because of ongoing degeneration for years prior to diagnosis. Studying the early consequences of dopamine loss is critical to understanding mechanisms of dopamine depletion disease progression and identifying biomarkers for earlier diagnosis so that effective treatment can ensue. Here, we created a model of dopamine loss in adult rats to study early transcriptional changes following loss of dopamine synthesis. We used CRISPR to knock out tyrosine hydroxylase (TH), the rate-limiting enzyme for dopamine synthesis, in the adult rat midbrain through injection of Th guide RNAs into transgenic rats expressing Cas9 nuclease specifically in dopamine transporter positive neurons and performed single nucleus RNA sequencing on midbrain and dorsal striatal tissue. Two weeks after injection, we measured a 90% loss of TH and dopamine in the midbrain, but striatal TH and dopamine were unaffected. Despite preceding striatal dopamine loss, our data identified alterations to genes involved in synaptic signaling, organization, and function in both midbrain and striatal neurons. We found the neurotrophic factor, neuregulin1, to be significantly reduced in dopaminergic neurons, which may account for transcriptional changes in surrounding cells that precede detectable reductions to striatal dopamine. These data establish a new rat model of adult TH loss and identify early transcriptional consequences of dopamine loss in the adult midbrain after normal development, providing potential biomarkers for early-stage dopamine loss in PD, synucleinopathies and related disorders of dopamine depletion.

neuroscience↗

bioRxiv2025

Generation and characterization of a tamoxifen-inducible, Cre driver rat for transgene expression in microglia

Microglia are the resident immune cells of the central nervous system (CNS) and display diverse functions under both physiological and pathological conditions. The past decade has seen burgeoning interest in microglia function, with a variety of transgenic tools developed for specific genetic manipulation of microglia in various injury, disease, and developmental models. Although the majority of models have been developed in mice, the ability to manipulate microglia in rats provides additional advantages to studying microglial function in the brain especially related to complex behavior. Using BAC transgenesis, our lab has created a transgenic rat (Cx3cr1-CreERT2) that expresses a tamoxifen inducible Cre recombinase (CreERT2) under control of the microglial/macrophage specific fractalkine C-X3-C Motif Chemokine Receptor 1 (Cx3cr1) promoter. In mice, CreERT2 and other transgenes have been expressed in microglia using the Cx3cr1 promoter, however, this is the first demonstration in rats. Importantly, these rats exhibit similar cognitive behaviors compared to their wildtype (WT) controls. Microglial specificity of inducible Cre expression was confirmed by breeding the novel Cx3cr1-CreERT2+/- rat with a previously reported double floxed inverse open reading frame (DIO)-mCherry+/- reporter rat to show tamoxifen inducible mCherry expression that colocalizes with the microglial marker Iba1. In addition, we utilize flow cytometry to demonstrate time and Cre dependent differences in recombination of Cx3cr1+ cells in the spleen, peripheral blood, and brain at two- and eight-weeks post-tamoxifen treatment. Overall, we have created a novel transgenic rat model for researchers to employ in understanding microglial and peripheral immune cell function in rats.

neuroscience↗

bioRxiv2025

SCOPE: Revealing Hidden Mechanisms in Phenotypic Screens Through Target and Pathway Enrichment

Phenotypic screening enables discovery of small molecules without requiring predefined targets, but mechanistic interpretation remains challenging due to polypharmacology and pathway complexity. We developed SCOPE (Screening Compound Ontology for Pathway Enrichment), a KNIME-based computational framework that resolves the molecular drivers of phenotypic activity by linking compound-level screening data to annotated targets and pathways. SCOPE integrates multi-source target annotations and performs statistical enrichment to identify shared mechanisms of action. Applied to a high-throughput screen for modulators of ER-stress induced secretion of endoplasmic reticulum (ER) resident proteins, a process known as exodosis, SCOPE identified calcium signaling as the most enriched KEGG pathway without prior biological context. Target enrichment revealed G protein-coupled receptors (GPCRs) involved in inositol 1,4,5-trisphosphate receptors (IP3Rs)-mediated signaling, with widespread antagonism among hit compounds implicating this pathway in the regulation of exodosis. Notably, SCOPE uncovered a novel role for the histamine receptor HRH1, which was validated by RNAi knockdown and pharmacological inhibition, implicating HRH1 as a potential therapeutic target in ER stress-related disorders. These results highlight SCOPEs potential to deconvolute phenotypic screens and uncover actionable mechanisms in complex cellular systems.

bioinformatics↗

bioRxiv2025

Experimental change in personality: Overexpression of GDNF in the rat striatum converts the low exploratory phenotype into highly explorative

Major vulnerability factors for psychiatric disorders such as depression, that often prevent complete remission and lead to relapses, are temperamental. In a rat model of clustered persistent high anxiety/low motivation, we have found that overexpression of glial-cell-line-derived neurotrophic factor (GDNF) by intra-striatally administered adeno-associated virus vector strikingly converts the passive coping style of low exploratory rats into an active one, similar to high exploratory rats. This conversion of behavioural strategy developed gradually over repeated testing, and was associated with increased catecholamine metabolism in several brain regions and changes in the regulation of serotonin neurotransmission. An increase in in vivo dopamine transporter availability in the striatum was necessary for the phenotype conversion. Associated changes in striatal gene expression included key players in monoamine storage and epitranscriptomic regulation. The increase in GDNF signalling also caused alterations in levels and regional covariation of oxidative metabolism, indicative of persistent reorganization of neural activity throughout the brain. Thus, neurotrophic factors, GDNF in particular, may play a pivotal role in the development, persistence and alteration of personality traits, and therefore constitute a potential target for treatment of chronic, relapsing psychiatric disorders.

neuroscience↗

bioRxiv2024

A Rapid Method for Producing Adeno-Associated Viral Vectors Suitable for Transducing Rodent Neurons in vitro and in vivo

The use of adeno-associated viral vectors for delivery of genetic information into the mammalian CNS remains popular but producing highly purified vectors for in vivo applications requires a significant investment of resources and time that can impede the development and testing of AAV vectors for experimentation. To address this issue, we have developed a simplified AAV packaging protocol that does not require large capital equipment (ultracentrifugation or chromatography machines) yet still produces virus in quantities that are sufficient for testing AAV prototypes in the rodent CNS. This protocol is serotype agnostic, and has been successful with AAV1, AAV9, AAV-DJ, and rAAV2-retro. Intracranial injection of AAV-EF1a-GFP-KASH into rats demonstrated that our "small scale" AAV preps produce patterns of transgene expression and inflammation that are similar to those produced by the same AAV vector purified by affinity column chromatography. Our protocol allows for multiple vectors to be packaged and processed in parallel, making it ideal for testing multiple variants, constructs, and prototypes simultaneously.

molecular biology↗

bioRxiv2023

Excitotoxic glutamate levels cause the secretion of resident endoplasmic reticulum proteins

Dysregulation of synaptic glutamate levels can lead to excitotoxicity such as that observed in stroke, traumatic brain injury, and epilepsy. The role of increased intracellular calcium (Ca2+) in the development of excitotoxicity is well established. However, less is known regarding the impact of glutamate on endoplasmic reticulum (ER)-Ca2+-mediated processes such as proteostasis. To investigate this, we expressed a secreted ER Ca2+ modulated protein (SERCaMP) in primary cortical neurons to monitor exodosis, a phenomenon whereby ER calcium depletion causes the secretion of ER resident proteins that perform essential functions to the ER and the cell. Activation of glutamatergic receptors (GluRs) led to an increase in SERCaMP secretion indicating that normally ER resident proteins are being secreted in a manner consistent with ER Ca2+ depletion. Antagonism of ER Ca2+ channels attenuated the effects of glutamate and GluR agonists on SERCaMP release. We also demonstrate that endogenous proteins containing an ER retention sequence (ERS) are secreted in response to GluR activation supporting that neuronal activation by glutamate promotes ER exodosis. Ectopic expression of KDEL receptors attenuated the secretion of ERS-containing proteins caused by GluR agonists. Taken together, our data indicate that excessive GluR activation causes disruption of neuronal proteostasis by triggering the secretion of ER resident proteins through ER Ca2+ depletion and describes a new facet of excitotoxicity. SignificanceDuring excitotoxicity, the excessive activation of glutamate receptors causes elevated intracellular calcium (Ca2+) that promotes cellular dysfunction and death. While the role of cytosolic Ca2+ in excitotoxicity has been well-studied, the consequences of changes in endoplasmic reticulum (ER) Ca2+ during excitotoxicity remains unclear. The relatively high concentration of calcium in the ER is necessary for ER resident proteins to function prop out essential functions and maintain cellular proteostasis. We show here that excitotoxic conditions destabilize the ER proteome by triggering ER resident protein secretion. Stabilizing ER Ca2+ or overexpressing receptors that interact with ER resident proteins can prevent disruption of proteostasis associated with excitotoxicity. The present study provides a new link between excitotoxicity, ER Ca2+ homeostasis, and the ER proteome.

neuroscience↗

bioRxiv2022

Effect of selective lesions of nucleus accumbens μ-opioid receptor-expressing cells on heroin self-administration in male and female rats: a study with novel Oprm1-Cre knock-in rats

The brain {micro}-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs. However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-based Oprm1-Cre knock-in transgenic rat that provides cell-type specific genetic access to MOR-expressing cells. After performing anatomical and behavioral validation experiments, we used the Oprm1-Cre knock-in rats to study the role of nucleus accumbens (NAc) MOR-expressing cells in heroin self-administration in male and female rats. Using RNAscope, autoradiography, and fluorescence in situ hybridization chain reaction (HCR-FISH), we found no differences in Oprm1 expression in NAc, dorsal striatum (DS), and dorsal hippocampus, or MOR receptor density (except DS) or function between Oprm1-Cre knock-in rats and wildtype littermates. HCR-FISH assay showed that iCre is highly co-expressed with Oprm1 (95-98%). There were no genotype differences in pain responses, morphine analgesia and tolerance, heroin self-administration, and relapse-related behaviors. We used the Cre-dependent vector AAV1-EF1a-Flex-taCasp3-TEVP to lesion NAc MOR-expressing cells and report sex-specific effects: the lesions decreased acquisition of heroin self-administration in male Oprm1-Cre rats and had a stronger inhibitory effect on the effort to self-administer heroin in female Oprm1-Cre rats. The validation of an Oprm1-Cre knock-in rat enables new strategies for understanding the role of MOR-expressing cells in rat models of opioid addiction, pain-related behaviors, and other opioid-mediated functions. Our initial mechanistic study with these rats suggests a sex-specific role of NAc MOR-expressing cells in heroin self-administration. Significance statementThe brain {micro}-opioid receptor (MOR) is critical for the analgesic, rewarding, and addictive effects of opioid drugs. However, in rat models of opioid-related behaviors, the circuit mechanisms of MOR-expressing cells are less known because of a lack of genetic tools to selectively manipulate them. We introduce a CRISPR-based Oprm1-Cre knock-in transgenic rat that provides cell-type specific genetic access to brain MOR-expressing cells. After performing anatomical and behavioral validation experiments, we used the Oprm1-Cre knock-in rats to show a potential sex-specific role of nucleus accumbens MOR-expressing cells in heroin self-administration. The new Oprm1-Cre rats can be used to study both the general and sex-specific role of brain MOR-expressing cells in animal models of opioid addiction, pain-related behaviors, and other opioid-mediated functions.

neuroscience↗

bioRxiv2022

Caffeine and MDMA (ecstasy) exacerbate ER stress triggered by hyperthermia

Drugs of abuse can cause local and systemic hyperthermia, a known trigger of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Another trigger of ER stress and UPR is ER calcium depletion which causes ER exodosis, the secretion of ER resident proteins. Club drugs such as 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) can create hyperthermic conditions in the brain and cause toxicity that is affected by the environmental temperature and the presence of other drugs, such as caffeine. Here we examine the secretion of ER resident proteins and activation of the UPR under combined exposure to MDMA and caffeine in a cellular model of hyperthermia. We show that hyperthermia triggers the secretion of normally ER resident proteins and that this aberrant protein secretion is potentiated by the presence of MDMA, caffeine, or a combination of the two drugs. Hyperthermia activates the UPR but the addition of MDMA or caffeine does not alter canonical UPR gene expression despite the drug effects on ER exodosis of UPR-related proteins. One exception was increased BiP/Grp78 mRNA levels in MDMA-treated cells exposed to hyperthermia. These findings suggest that club drug use under hyperthermic conditions exacerbates disruption of ER proteostasis contributing to cellular toxicity. HighlightsO_LIER resident proteins are redistributed into the extracellular space in response to hyperthermia and caffeine and MDMA further enhance this secretion. C_LIO_LIStabilizing ER calcium and overexpressing KDEL receptors reduces ER resident protein secretion following hyperthermia. C_LIO_LIHyperthermia triggers a UPR response with MDMA augmenting BiP expression in hyperthermic conditions. C_LI

cell biology↗

bioRxiv2021

Identification of ER/SR resident proteins as biomarkers for ER/SR calcium depletion in skeletal muscle cells

Aberrations to endoplasmic/sarcoplasmic reticulum (ER/SR) calcium concentration can result in the departure of endogenous proteins in a phenomenon termed exodosis. Redistribution of the ER/SR proteome can have deleterious effects to cell function and cell viability, often contributing to disease pathogenesis. Many proteins prone to exodosis reside in the ER/SR via an ER retention/retrieval sequence (ERS) and are involved in protein folding, protein modification, and protein trafficking. While the consequences of their extracellular presence have yet to be fully delineated, the proteins that have undergone exodosis may be useful for biomarker development. Skeletal muscle cells rely upon tightly coordinated ER/SR calcium release for muscle contractions, and perturbations to calcium homeostasis can result in myopathies. Ryanodine receptor type-1 (RYR1) is a calcium release channel located in the SR. Mutations to the RYR1 gene can compromise calcium homeostasis leading to a vast range of clinical phenotypes that include hypotonia, myalgia, respiratory insufficiency, ophthalmoplegia, fatigue and malignant hyperthermia (MH). There are currently no FDA approved treatments for RYR1-related myopathies (RYR1-RM). Here we examine the exodosis profile of skeletal muscle cells following ER/SR calcium depletion. Proteomic analysis identified 4,465 extracellular proteins following ER/SR calcium depletion with 1,280 proteins significantly different than vehicle. A total of 54 ERS proteins were identified and 33 ERS proteins significantly increased following ER/SR calcium depletion. Specifically, ERS protein, mesencephalic astrocyte-derived neurotrophic factor (MANF), was elevated following calcium depletion, making it a potential biomarker candidate for human samples. Despite no significant elevation of MANF in plasma levels among healthy volunteers and RYR1-RM individuals, MANF plasma levels positively correlated with age in RYR1-RM individuals, presenting a potential biomarker of disease progression. Selenoprotein N (SEPN1) was also detected only in extracellular samples following ER/SR calcium depletion. This protein is integral to calcium handling and SEPN1 variants have a causal role in SEPN1-related myopathies (SEPN1-RM). Extracellular presence of ER/SR membrane proteins may provide new insight into proteomic alterations extending beyond ERS proteins. Pre-treatment of skeletal muscle cells with bromocriptine, an FDA approved drug recently found to have anti-exodosis effects, curbed exodosis of ER/SR resident proteins. Changes to the extracellular content caused by intracellular calcium dysregulation presents an opportunity for biomarker development and drug discovery.

cell biology↗

bioRxiv2021

Computational modeling of C-terminal tails to predict the calcium-dependent secretion of ER resident proteins

The lumen of the endoplasmic reticulum (ER) has resident proteins that are critical to perform the various tasks of the ER such as protein maturation and lipid metabolism. These ER resident proteins typically have a carboxy-terminal ER retention sequence (ERS). The canonical ERS is Lys-Asp-Glu-Leu (KDEL) and when an ER resident protein moves from the ER to the Golgi, KDEL receptors (KDELRs) in the Golgi recognize the ERS and return the protein to the ER lumen. Depletion of ER calcium leads to the mass departure of ER resident proteins in a process termed exodosis, which is also regulated by KDELRs. Here, by combining computational prediction with machine learning-based models and experimental validation, we identify carboxy tail sequences of ER resident proteins divergent from the canonical "KDEL" ERS. Using molecular modeling and simulations, we demonstrated that two representative non-canonical ERS can stably bind to the KDELR. Collectively, we developed a method to predict whether a carboxy-terminal sequence acts as a putative ERS that would undergo secretion in response to ER calcium depletion and interact with the KDELRs. Identification of proteins that undergo exodosis will further our understanding of changes in ER proteostasis under physiological and pathological conditions where ER calcium is depleted.

cell biology↗