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Age-dependent Protection and Vulnerability to Drugs of Abuse in Nonhuman Primates

John Elsworth, Yale University

Overview: The addictive properties of methamphetamine (METH) rely on its interaction with dopamine (DA) neurons where it provokes increased dopaminergic transmission in the brain’s reward system. METH also damages DA neurons, leading to long-lasting health implications in users, such as increasing the risk for developing Parkinson’s disease. Dopaminergic toxicity may also be a contributory driver of METH dependence, as a strong negative correlation exists in users between the extent of DA dysfunction and the ability to maintain sobriety and even following prolonged abstinence, former users exhibit cognitive decline and impaired inhibitory control, deficits that implicate dopaminergic dysregulation. In a nonhuman primate (NHP) model, we have found that the neurotoxic effect of METH on DA neurons is crucially dependent on the monkey’s age at the time of exposure. Very early in development these neurons are protected by unknown biochemical mechanisms that prevent the toxic effect of METH typical seen in adults, even when young NHP are exposed to higher circulating levels of METH than older subjects. If these protective factors can be identified and their properties verified, then pharmacological or viral vector-induced reinstatement of their expression later in life offers a strategy to repair and/or protect dopamine neurons in METH users and others at risk for striatal DA deficiency.

In the past year we have executed two studies with African green monkeys at our NHP facility in St Kitts (West Indies) to study these developmental protective factors. These monkeys are plentiful in the island, free from all known pathogens and relatively inbred ensuring relatively low variability between samples. One study involved collection of tissue from untreated age-matched groups of young and adult, male and female, NHPs. While our qPCR studies have revealed two candidate neuroprotective genes that are highly expressed early in life in dopaminergic regions, proteomic profiling of our collected tissue will reveal other promising targets with antioxidant properties and important information about their regulatory pathways.

The anti-diabetic drug, metformin, is known to have neuroprotective actions. Metformin is classified as an activator of AMP-activated protein kinase (AMPK), which plays a key role regulating cellular energy homeostasis, but there is evidence now that its neuroprotective properties do not rely on AMPK activation. We have examined the impact of chronic metformin on METH-induced changes to the dopaminergic system in the NHP brain. Interestingly we did observe protection of dopamine neurons in the absence of AMPK activation, so an in-depth proteomic profiling of collected tissues should provide valuable candidate compounds connected with its neuroprotective properties that could be utilized to design more efficient strategies to protect primate dopamine neurons. To this end, the Center has so far generated libraries from 4 brain regions (SN, VTA, STR, DFC) by LC-MS/MS, and detected ~4700-4800 proteins in each region. To identify pathways activated by metformin, they performed TiO2 enrichment on desalted tryptic peptides followed by DIA LC-MS/MS analysis on samples of SN both before and after enrichment, which revealed proteins that were significantly increased phosphorylation. Analyses are on-going and are being performed in Scaffold, Qlucore, and Cytoscape software. Recently, African green monkey data were added to the ClueGO repository, a Cytoscape App that can perform Gene Ontology (GO) analyses for our unique model. GO terms associated with proteins down-regulated by METH include ‘mitochondrial transcription’, ‘metabolic process’, and ‘dopaminergic synapse’. Metformin increased phosphorylation of 26 proteins, including DARPP-32, and associated GO terms included ‘mitochondrial fission’ and ‘protein targeting to mitochondrion’.

Proposed Research: Adolescence is a highly vulnerable period for the developing brain, when the connectivity between GABA, glutamate and dopamine neurons in the prefrontal cortex are being established. THC targets these neuronal systems and emerging evidence indicates that adolescent exposure to THC is associated with a high risk of developing psychiatric diseases, suggesting that disturbance of prefrontal cortex maturation by THC contributes to its propensity to induce schizophrenia-like signs. Our planned study will compare the dorsolateral prefrontal cortex of adolescent and adult monkeys following administration of THC or vehicle using the proteomics approach described above. We expect that these data will reveal novel insights into the differential vulnerability of the adolescent brain to THC and suggest approaches for protecting or treating the disturbances provoked by the drug at this vulnerable stage of primate life.