Heroin-addicted individuals have alterations in the expression a gene called FYN also known to regulate the production of Tau

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Heroin-addicted individuals have alterations in the expression a gene called FYN – a gene known to regulate the production of Tau, a protein that is highly elevated and implicated in neurocognitive disorders like Alzheimer’s disease.

The study emphasizes that opioid use can affect the brain in a way that might increase vulnerability of neural systems that trigger neurodegeneration later in life; however, since these changes are epigenetic (alterations in gene function that are influenced by environmental factors and not alterations of the DNA itself), they are reversible and medications that have already been developed to target FYN for neurodegenerative disorders may be studied as a novel treatment for opioid addiction.

Interestingly, findings were consistent across human, animal and cell models.

Through post-mortem analysis of the brains of human heroin users, the team found that, specifically in neurons, the most significantly impaired epigenetic region is related to a gene called FYN.

Essentially, heroin ‘opened up’ the DNA at the FYN gene, which encodes a protein called tyrosine kinase FYN, that is strongly linked to synaptic plasticity and which directly results in production of Tau.

Too much Tau in the brain is associated with neurodegenerative diseases. Researchers observed that expression and activity of tyrosine kinase FYN was also induced in rats trained to self-administer heroin and also in primary striatal neurons treated with chronic morphine in vitro.

Additionally, they demonstrated that inhibition of the FYN kinase (either via pharmacological means or through genetic manipulation) reduces heroin-seeking and heroin-taking behaviors.

The findings will increase awareness about the potential impact of heroin to alter neural systems related to neurodegenerative disorders.

The findings also identify FYN inhibitors as a novel therapeutic treatment for heroin use disorders.

Human brains from a cohort of subjects who succumbed to heroin overdose and normal controls, translational animal model of rats trained to self-administer heroin, and primary striatal neurons treated with chronic morphine in vitro were studied.

Adult animals were exposed to heroin and their brains later studied.

The researchers performed unbiased, cell-type-specific, genome-wide profiling of chromatin accessibility, providing insights into epigenetic regulation directly in the brains of heroin-addicted individuals.

To assess the causal relationship between heroin use and FYN pathology, they studied the brains of rats trained to self-administer heroin and they hit primary striatal neurons with chronic morphine in petri dishes to examine the effect at the individual cellular level.

By scanning the entire genome of heroin users to identify whether disturbances in how genes are turned on or off exist, Mount Sinai researchers found that heroin opened up the DNA at the FYN gene.

The FYN gene is known to regulate the production of Tau, a protein implicated in neurodegenerative disorder like Alzheimer’s disease, meaning that heroin may put users at an increased risk of neurodegenerative disease later in life.

Importantly, these novel findings suggest that FYN inhibitors (which have already been developed and are being assessed for use in Alzheimer’s disease) may be promising therapeutic tools for heroin-use disorder.

Said Mount Sinai’s Dr. Yasmin Hurd of the research: “Drug overdoses due to opioid abuse remain at epidemic levels and continue to rise precipitously during the current pandemic, with novel treatments desperately needed.

Direct molecular insights into the heroin-addicted human brain are critical to guide future therapies. Our new study findings clearly open up new lines of treatment opportunities for opioid use disorder, which could benefit and potentially save the lives of so many.”


Accumulating evidence shows that exposure to cocaine or heroin influences the function of several tyrosine kinases (Lee and Messing 2008; Nestler 1994) which are involved in the regulation of transduction mechanisms both in neurons and non-neuronal populations, thereby contributing to the regulation of synaptic homeostasis and plasticity, as well as inflammation.

In neurons, the src kinases, such as Fyn and Lyn, control synaptic mechanisms, including plasticity, downstream of the NMDA receptor (Hayashi et al. 1999). Fyn is involved in the cocaine-induced alteration of NMDA-mediated glutamatergic transmission in the ventral tegmental area or the dorsal hippocampus that underlies the sensitisation to the psychomotor properties of cocaine (Schumann et al. 2009) or context-induced reinstatement of an extinguished instrumental response for cocaine (Xie et al. 2013), respectively.

Similarly, src kinases have been suggested functionally to converge with PKC in the regulation of NMDA receptors by μ-opiate receptors (Garzon et al. 2008), the phosphorylation of which by Fyn is also involved in heroin withdrawal (Zhang et al. 2017).

In non-neuronal populations, cocaine and heroin trigger the src kinase-dependent activation of microglia and astrocytes in the brain and the activation of mast cells (Galli et al. 1993), the latter being involved in the propagation of drug-induced peripheral inflammation (de Timary et al. 2017; Liang et al. 2016; Nevidimova et al. 2015) to the brain (Kousik et al. 2012; Lacagnina et al. 2017).

Exposure to alcohol, cocaine and heroin triggers activation of astrocytes and microglia (Miguel-Hidalgo 2009), especially in the striatum where astrocytes, through their regulation of glutamate homeostasis, have been shown in rats to play a pivotal role in the propensity to reinstate extinguished instrumental responding for cocaine and heroin (Knackstedt and Kalivas 2009; Scofield and Kalivas 2014) and compulsive relapse after escalated intake of cocaine (Ducret et al. 2016).

The involvement of astrocytes in mediating the reinforcing effect of cocaine has been recently shown to be under the control of inflammatory processes (Northcutt et al. 2015; but see Skolnick et al. 2014 for further discussion), the systemic activation of which has also been suggested to increase striatal dopamine release in response to stimulant drugs (Petrulli et al. 2017).

The primary mechanism bridging peripheral and central inflammation relies on the c-kit tyrosine kinase-dependent activation of peripheral mast cells (Dubreuil et al. 2009) and their subsequent degranulation in the brain (Dong et al. 2017; Skaper et al. 2014; Zhang et al. 2016).

Even though there are resident mast cells in the brain (Zhuang et al. 1996), which provide up to 50 % of the brain’s histamine (Goldschmidt et al. 1985), the activation of glial cells (Skaper et al. 2014) and associated neuroinflammation depends on mast cells from the periphery (Dong et al. 2014; Theoharides 1990).

Following activation, mast cells from the periphery have the ability to cross the blood brain barrier (Nautiyal et al. 2008), the permeability of which they control (Zhuang et al. 1996), and rapidly invade the brain where, upon degranulation, they release mediators such as dopamine, serotonin and CRF alongside cytokines and histamine (Dong et al. 2014).

Thus, by triggering c-kit and Fyn-dependent activation and degranulation of mast cells, cocaine and heroin influence the permeability of the BBR and facilitate the release of histamine (Brown et al. 2001; Di Bello et al. 1998; Mannaioni et al. 1996).

This degranulation-induced histamine release may activate astrocytes by recruiting cAMP-dependent signalling (Agullo et al. 1990) and independently influence histaminergic control of the function of the mesolimbic dopamine system, thereby directly interacting with the reinforcing or incentive properties of addictive drugs (Banks et al. 2009; Brabant et al. 2006, 2009; Ellenbroek 2013; Masukawa et al. 1993).

Thus, the tyrosine kinases c-kit and Fyn, alongside other members of the src kinase family, play a major role in the within- and between-systems adaptations to chronic exposure to cocaine or heroin that may influence the development of addiction. However, the lack of well-tolerated selective inhibitors with limited side effects has hitherto prevented the investigation of the influence of their chronic inhibition on the reinforcing and incentive properties of cocaine and heroin.

Masitinib is an oral active tyrosine kinase inhibitor that potently targets a limited number of kinases including c-Kit, Fyn and Lyn, as well as platelet-derived growth factor receptors, thereby controlling the central effects of Fyn and Lyn, the permeability of the BBR and the activation and degranulation of mast cells (Dubreuil et al. 2009).

Studies involving kinase inhibitor selectivity have shown that masitinib is one of the most selective kinase inhibitors under development (Anastassiadis et al. 2011), thereby limiting the potential for off-target effects. Accordingly, masitinib has been shown to be effective and safe to use in humans for the treatment of mast cell-related diseases such as severe mastocytosis (Lortholary et al. 2017), severe refractory asthma (Humbert et al. 2009) and rheumatoid arthritis (Tebib et al. 2009), as well as in stroke (Gagalo et al. 2015), Alzheimer’s disease (Piette et al. 2011), multiple sclerosis (Vermersch et al. 2012) and depression (Moura et al. 2011, 2012).

We therefore investigated the influence of chronic daily per os administration of masitinib on the reinforcing and motivational properties of cocaine (250 μg/infusion) and heroin (40 μg/infusion) as compared to food. Three different cohorts of rats were trained to self-administer either cocaine, heroin or food and challenged under specific behavioural conditions to assess the influence of chronic masitinib administration on their sensitivity to the reinforcing properties of, and their motivation for, the drugs, as well as their propensity to relapse.

Discussion
The results of the present study demonstrate that daily treatment with the tyrosine kinase inhibitor masitinib results in a robust decrease in the reinforcing and motivational properties of cocaine in male rats with a relatively long history of self-administration of a unit dose of 250 μg/infusion.

Thus, masitinib decreased cocaine intake under continuous reinforcement, prevented an increase in responding in the face of increasing behavioural demand for cocaine (Salamone et al. 2003) and decreased the break point under a progressive ratio schedule of reinforcement.

This decrease in the motivation for cocaine in masitinib-treated rats was further supported by a marked decrease in the persistence of responding under extinction and the propensity to relapse after forced abstinence.

The effect of masitinib on instrumental responding was highly specific to cocaine at the dose tested as the same treatment had no effect on the reinforcing and motivational effects of food and heroin at the dose of 40 μg/infusion. The absence of effect on the motivation for food is in agreement with the overall lack of effect of masitinib in humans on feeding or general motivation.

Thus, masitinib was recently shown to have potential therapeutic effects in depression in patients with mastocytosis (Moura et al. 2011, 2012), as well as in Alzheimer’s disease (Piette et al. 2011) and multiple sclerosis in humans (Vermersch et al. 2012), thereby demonstrating that this drug is safe to use.

Much to our surprise, masitinib had no effect on the reinforcing and motivational properties of heroin, suggesting that selective inhibition of c-kit, Fyn and Lyn exerted in the nanomolar range by masitinib only impinged on brain mechanisms of reinforcement engaged by cocaine in male rats.

In the light of the available research, it is difficult to put forward a definitive mechanism of the effect of masitinib. However, the specific behavioural effects and putative therapeutic potential of masitinib reported here warrant further research on the cellular mechanisms by which it exerts these effects.

Even though masitinib has high affinity for the microglial factor CSFR1, involved in microglia survival and activation, the differential influence of masitinib on the reinforcing and incentive properties of cocaine and heroin is very unlikely to be accounted for by a direct influence on central inflammatory mechanisms.

Indeed, N-acetylcysteine (NAC) (Murray et al. 2012a), which acts centrally to prevent drug-induced neuroinflammation (Schneider et al. 2017) and directly targets astrocytes (Olive et al. 2012), does not show cocaine-specific effects as it decreases both cocaine and heroin-seeking behaviour (Hodebourg et al. 2018; Murray et al. 2012b). The specificity of the effects of masitinib is therefore more likely related to its effects on c-Kit, Fyn and Lyn.

Since neuronal Fyn and Lyn are involved in the regulation of NMDA-dependent synaptic mechanisms influenced by addictive drugs, including cocaine, alcohol and heroin (Ge et al. 2017; Schumann et al. 2009; Wang et al. 2010; Yaka et al. 2002), a direct influence of masitinib on Fyn may be a promising candidate mechanism.

However, intracerebral inhibition of Fyn by the src kinase antagonist PP2 has been shown to inhibit heroin seeking as measured in a context-induced reinstatement procedure (Ge et al. 2017). Therefore, even if a direct influence on neuronal mechanisms cannot be ruled out, it seems unlikely to account for the differential effect of masitinib on the reinforcing and motivational properties of cocaine and heroin observed here.

However, Fyn and Lyn, alongside c-kit, primarily control the activation, migration and degranulation of mast cells, and therefore the mast cell glia axis (Zhang et al. 2016). Indeed, at the dose tested, masitinib is particularly efficient at inhibiting mast cells, thereby preventing drug-induced recruitment of neuroinflammatory mechanisms from the periphery (Di Bello et al. 1998; Dong et al. 2014; Petrulli et al. 2017; Silverman et al. 2000) and protecting against cocaine-induced alteration of the blood brain barrier (Kumar 2011; Sharma et al. 2009), the permeability of which is also controlled by mast cells (Esposito et al. 2002; Zhuang et al. 1996).

However, the nature of the behavioural response to masitinib suggests it does not influence the brain availabilty of cocaine or heroin as such a difference would result in a upward vertical shift in responding under continuous reinforcement.

One potential alternative mechanism is related to the prevention by masitinib of drug-induced degranulation of activated mast cells having entered the brain. Thus, upon activation, these multifunctional cells enter the brain where alongside resident mast cells primarily, but not exclusively located in the thalamus (Dimitriadou et al. 1990; Goldschmidt et al. 1985; Zhuang et al. 1996), they release cytokines and neuromediators in their microenvironment.

The neuromediators released by activated mast cells in the brain, include dopamine (in particular in the mesolimbic system; (Dropp 1976), corticotropin releasing factor, serotonin and histamine (Goldschmidt et al. 1985; Ronnberg et al. 2012a, b), which have all been shown to influence cocaine reinforcement (Silverman et al. 2000).

Histamine is of particular interest for the selectivity of the effects of masitinib on the motivational properties of cocaine as histaminergic mechanisms that influence the mesostriatal dopamine system (Banks et al. 2009; Ellenbroek 2013; Tanda et al. 2008).

Although the nature of the interaction between the histamine and the dopamine system and consequent modulation of the reinforcing effects of cocaine remain to be elucidated (Banks et al. 2009; Brabant et al. 2009; Holtz et al. 2013; Ito et al. 1997; Oleson et al. 2012), histamine has been shown to act on different neuronal systems either to inhibit or activate midbrain dopamine activity (Fleckenstein et al. 1993; Molina-Hernandez et al. 2000; Schlicker et al. 1993).

Dopaminergic transmission in the mesolimbic system is a key mechanism underlying the reinforcing effects of cocaine, but less so heroin (Ettenberg et al. 1982; Pettit et al. 1984). Thus, heroin self-administration is unaffected by dopamine depletion from the nucleus accumbens, which in marked contrast reduces cocaine self-administration and progressive ratio break points for the drug (for review see Badiani et al. 2011).

Since histamine brain levels are much more influenced by mast cells than the widespread projections from tuberomamillary nucleus of the hypothalamus, by inhibiting the degranulation of mast cells, masitinib may alter histaminergic control of dopaminergic mechanisms that underlie the reinforcing and motivational effects of cocaine, but not heroin.

This clearly indicates the need for further investigation of the cellular mechanisms that mediate the effects of masitinib-induced degranulation of mast cells on the motivational effects of cocaine.

Additionally, in order fully to characterise the influence of masitinib on the reinforcing and motivational properties of cocaine, further investigations are required to test whether the effects observed here for the unit dose of 250 μg/infusion are generalised across a range of doses.

Similarly, whether the decreased propensity instrumentally to respond under extinction after 10 days of forced abstinence results from a long-lasting influence of masitinib on previous cocaine self-administration or a direct effect on the latter should be further investigated.

Nevertheless, the present results suggest that a novel highly selective tyrosine kinase inhibitor that primarily targets mast cells activation and safe to use in humans decreases the reinforcing and motivational properties of 250 μg/infusion cocaine.

reference link : https://europepmc.org/article/PMC/5920000


More information: Gabor Egervari et al, Chromatin accessibility mapping of the striatum identifies tyrosine kinase FYN as a therapeutic target for heroin use disorder, Nature Communications (2020). DOI: 10.1038/s41467-020-18114-3

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