Small molecules are important tools to measure and modulate intracellular signaling pathways. A longstanding limitation for using chemical compounds in complex tissues has been the inability to target bioactive small molecules to a specific cell class. Here, we describe a generalizable esterase-ester pair capable of targeted delivery of small molecules to living cells and tissue with cellular specificity. We used fluorogenic molecules to rapidly identify a small ester masking motif that is stable to endogenous esterases, but is efficiently removed by an exogenous esterase. This strategy allows facile targeting of dyes and drugs in complex biological environments to label specific cell types, illuminate gap junction connectivity, and pharmacologically perturb distinct subsets of cells. We expect this approach to have general utility for the specific delivery of many small molecules to defined cellular populations. cellular imaging | microscopy | enzyme substrates | fluorophores | pharmacological agents C hemical probes are essential tools in biology for measuring and manipulating cellular properties. Optimization of the structural and electronic features of small molecules allows the fine-tuning of molecular recognition specificity for a particular cellular target. Even with high molecular specificity, however, the application of small molecules in complex biological environments is frequently limited by poor cellular specificity. The inability to target small molecules, such as imaging or pharmacological agents, to defined cellular populations can confound the evaluation and control of discrete subsets of cells within a multicellular environment. A general and efficient strategy for cell-specific targeting, combining the molecular specificity of small molecules with the cellular specificity of genetics, would allow intracellular pathways in defined cell types to be selectively probed in complex tissues.An attractive approach for general cell-specific delivery of small molecules employs selective enzyme-substrate pairs. In this strategy, compounds are masked by attachment of a standard, disposable blocking group that is stable to native cellular enzymes, but labile to a specific exogenous enzyme. Expression of such a protein in a genetically defined cell population permits unmasking of the small molecule with cellular specificity. To be useful across experimental paradigms, such a system should utilize an enzyme that unmasks molecules with high efficiency, expresses in different cell types, and exhibits low cellular toxicity. The cognate masking group must be modular, synthetically efficient, and allow molecules to diffuse passively across the cellular membrane, while also exhibiting favorable solubility and stability in aqueous solution.To date only a few enzyme-substrate pairs have been used as targeted delivery systems for small molecules, and none meet all the criteria outlined above. Strategies employing enzymes encoded by common reporter genes (1) have found some success in targeting small molecules (2-4), ...

Objective Nigrostriatal reserve refers to the threshold of neuronal injury to dopaminergic cell bodies and their terminal fields required to produce parkinsonian motor deficits. Inferential studies have estimated striatal dopamine reserve to be at least 70%. Knowledge of this threshold is critical for planning interventions to prevent symptom onset or reverse nigrostriatal injury sufficient to restore function in people with Parkinson disease. In this study, we determine the nigrostriatal reserve in a non-human primate model that mimics the motor manifestations of Parkinson disease. Methods Fifteen macaque monkeys received unilateral randomized doses of the selective dopaminergic neuronal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. We compared blinded validated ratings of parkinsonism to in vitro measures of striatal dopamine and unbiased stereologic counts of nigral neurons after tyrosine hydroxylase immunostaining. Results The percent of residual cell counts in lesioned nigra correlated linearly with the parkinsonism score at 2 months (r = −0.87, p <0.0001). The parkinsonism score at 2 months correlated linearly with the percent residual striatal dopamine (r = −0.77, p = 0.016) followed by a flooring effect once nigral cell loss exceeded 50%. A reduction of about 14 to 23% of nigral neuron counts or 14 to 37% of striatal dopamine was sufficient to induce mild parkinsonism. Conclusions The nigral cell body and terminal field injury needed to produce parkinsonian motor manifestations may be much less than previously thought.

Apathy, primarily defined as a lack of motivation, commonly occurs in people with Parkinson disease (PD). Although dysfunction of basal ganglia pathways may contribute to apathy, the role of dopamine remains largely unknown. We investigated the role of dopaminergic pathways in the manifestation of apathetic behaviors by measuring the effects of the selective dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on monkeys’ willingness to attempt goal directed behaviors, distinct from their ability to perform tasks. Fifteen macaques received variable doses of MPTP, had PET scans with [11C]-dihydrotetrabenazine (DTBZ), [11C]-2β-3β-4-fluorophenyltropane (CFT), and [18F]-fluorodopa (FD) and performed tasks to assess apathetic behaviors and motor impairment. At 8 weeks post-MPTP, primates were euthanized and stereological cell counts and dopamine measurements were done. Apathy scores were compared to motor scores, in vitro and in vivo dopaminergic measures. Apathy scores increased following MPTP and correlated with DTBZ (rS = −0.85), CFT (rS = −0.87), and FD (rS = −0.85) specific uptake in nucleus accumbens (NAcc,) and dopaminergic cell counts in ventral tegmental area (VTA, rS = −0.80). Dopaminergic cell loss in VTA provided significant predictive power for apathy scores after controlling for the influence of cell loss in SN. Additionally, forward step-wise regression analyses indicated that neuropathological changes in the VTA-NAcc pathway predict apathetic behavior better than motor impairment or neuropathological changes in the nigrostriatal network. Our findings suggest that dopaminergic dysfunction within the VTA-NAcc pathway plays a role in the manifestation of apathetic behaviors in MPTP-lesioned primates. Similar changes in people with PD may contribute to apathy.