# Dopamine Transporter: Tools for in vivo molecular replacement

> **NIH NIH R21** · UNIV OF MASSACHUSETTS MED SCH WORCESTER · 2021 · $209,375

## Abstract

Dopamine (DA) is a major neurotransmitter with diverse physiological impact, and is required for movement,
sleep, working memory, and reward. Following evoked release, DA extracellular half-life is determined by
presynaptic reuptake, mediated by the SLC6 plasma membrane DA transporter (DAT). Addictive and
therapeutic psychostimulants, such as amphetamine, cocaine and methylphenidate (Ritalin), potently inhibit
DA uptake, sustain DA signaling and impact DA-dependent behaviors. DAT coding variants are implicated in a
variety of neuropsychiatric disorders, and transgenic mouse studies clearly demonstrate that DAergic signaling
and behaviors, as well as psychostimulant efficacy, are highly sensitive to the level of DAT expression. DAT is
not static at the plasma membrane, but is subject to robust constitutive and regulated endocytic recycling.
However, it remains unclear whether regulated DAT internalization impacts DAergic function and/or DA-dependent behaviors. Our central hypothesis is that regulated DAT trafficking is likely a critical and influential
determinant of DA signaling and DA-dependent behaviors. To test this hypothesis directly, we aim to replace
endogenous mouse DAT with trafficking dysregulated DAT mutants in adult mice. However, germline DAT
perturbations have clear developmental compensatory issues, underlining the need for a system that evaluates
DAT mutants in vivo, but circumvents the pitfalls of germline mutant expression. To this end, we have
developed a mouse model that integrates mouse with a floxed DAT gene (DATfl/fl) with a DAergic TET-OFF
mouse (Pitx3IRES2-tTA). The resulting DATfl/fl;Pitx3IRES2-tTA mouse will facilitate AAV-mediated mouse DAT
excision with Cre recombinase, and transgene replacement, driven by the Tet-responsive element, for in vivo
molecular replacement studies. We contend that this system offers considerable advantages over germline
knock-in approaches to test mutant protein function, as it circumvents developmental compensation and
additionally allows for circuit-specific replacement using AAVs. Feasibility for this approach is strongly
supported by strong preliminary data. The main aims of this two-year project are: 1) to optimize conditions for
in vivo DAT molecular replacement, and 2) to test whether DAT “gain-of-function” and “loss of function”
endocytic mutants impact DAT trafficking, DAergic signaling, and DA-dependent behaviors in male and female
mice. We anticipate that at the completion of these studies we will have developed a powerful mouse system
to interrogate the impact of DAT mutants in vivo in a manner that, heretofore, was not feasible. Moreover, we
expect that our new mouse model will have broad utility for a number of researchers aiming to evaluate mutant
function in adult mouse DAergic circuits.

## Key facts

- **NIH application ID:** 10133035
- **Project number:** 5R21DA049057-02
- **Recipient organization:** UNIV OF MASSACHUSETTS MED SCH WORCESTER
- **Principal Investigator:** Haley E Melikian
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $209,375
- **Award type:** 5
- **Project period:** 2020-04-01 → 2022-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10133035

## Citation

> US National Institutes of Health, RePORTER application 10133035, Dopamine Transporter: Tools for in vivo molecular replacement (5R21DA049057-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10133035. Licensed CC0.

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