Writeup | mGluR3 Enhancement and GCP-II Inhibition

Enhancing mGluR3 is one of the nootropic pathways with the highest potential. It has direct implications to do with modulating many mental disorders and functions, including hedonism, spatial cognition, verbal intelligence, motivation & more. This post will talk about enhancing mGluR3 and doing this through GCP-II inhibition, which has other benefits.

Introduction to mGluR3

Metabotropic glutamate receptor 3 (mGluR3/mGlu3) is part of a larger family of eight metabotropic receptors mGluR1-8 [1]. These eight receptors are classed generically into three groups, however the groups are not indicative of similar function (as discussed later). "Group I" contains mGluR1/5. "Group II" contains mGluR2/3. "Group III" contains mGluR4/6/7/8. However, through my research, mGluR3 is the most attractive receptor to modulate.

mGluR3 is a G_i/G₀-coupled G-protein coupled receptor (GPCR) generally localized to presynaptic sites of neurons in classical circuits. However, in higher cortical circuits (such as the dlPFC), mGluR3 are localized post-synaptically, where they strengthen rather than weaken synaptic connectivity. [1]

In layer III dlPFC, mGluR3 is found about 4/5 of the time postsynaptically, and 1/5 of the time presynaptically [2]. This is relevant because mGluR2 is located mostly presynaptically [7], and most studies use mixed mGluR2/3 ("Group II") modulators to make conclusions. This leads to weird mixed results with weird response curves as increasing mGluR2 inhibits cognitive function somewhat [8] (though decreasing it is not optimal either [3][4][5][6]), so using selective modulators of mGluR3 is much more desirable.

Stimulation of mGluR3 enhances the firing of Delay cells in the dlPFC and improves spatial tuning and working memory [2]. Delay cell firing is characterized by neural activity that persists during the retention interval of delay tasks, which is highly relevant for working memory and cognitive processes.

In a spatial working memory task, a delay cell will show sustained increased firing when an animal has to remember a particular spatial location over a delay. Delay cells are found in highest concentration in layer III of dlPFC, the main focus of working memory representations. [10]

mGluR3 Modulation is more desirable than mGluR2

Studies have shown inconsistent results with mGluR2 modulation [9], with some PAMs (such as BINA) increasing cognition in low-medium doses but then inhibiting function in high doses. Also in mGluR2 KO models, they have shown increased addictive tendancies [5][6], while increasing mGluR3 does not have these downsides.

mGluR2 is not simply the opposite of mGluR3. Its presynaptic location [7], differences in distribution [11][12] and actions on other pathways make it a less desirable pathway to modulate.

Using GCP-II -> NAAG to target mGluR3

GCP-II, also known as "NAAG Peptidase" is an enzyme that converts (catalyzes the hydrolysis) of NAAG to glutamate and NAA. Therefore, inhibiting GCP-II increases levels of NAAG. [34].

GCP-II Inhibition -> NAAG Enhancement

Due to low selectivity of most mGluR3 modulators, most studies around cognitive enhancement have used GCP-II inhibition instead of a mGluR3 PAM/agonist. This is because GCP-II -> NAAG only effects mGluR3, so it is inherently selective. As they are more proven (and also have benefits of cancer inhibition [47]), GCP-II inhibitors currently look more attractive than mGluR3 PAMs.

N-acetylaspartylglutamate (NAAG) is the third most prevalent and widely distributed transmitter/neuropeptide behind glutamate and GABA. It is co-expressed in neurons with several different primary amine transmitters, including glutamate and GABA. [13]

NAAG is co-released with these amine transmitters under conditions of elevated neuronal activity. Following release into the perisynaptic space NAAG activates the metabotropic receptor mGluR3 on post/presynaptic endings and glial cells. [13]

NAAG also effects NMDA (NR2A/B) to some degree depending on PH [14], but its effect on mGluR3 seems more significant.

NAAG does not effect mGluR2, instead it selectively activates mGluR3 [12]. This makes it superior to most mGluR3 PAMs because of the superior selectivity over mGluR2.

More on The Relevance of The dlPFC

The dorsolateral prefrontal cortex is the main area in which mGluR3 GCP-II inhibition enhances. The dlPFC is a major component [38] of motivation/anticipation and goals. The dorsolateral PFC (dlPFC) integrates and transmits signals of reward to the mesolimbic and meso-cortical DA circuits and initiates motivated behavior [39].

The dlPFC provides top-down regulation of emotion through indirect projections to BA25 via areas BA10m and BA32, and direct projections to BA24 [41]. The dlPFC interacts through BA10m (close to the PFC) and BA32 to eventually communicate with the amagdyla. The more newly evolved, rostral and lateral areas of PFC provide top-down regulation of the more primitive medial and caudal areas. The dlPFC is a key structure for executive and attentional control whereby any transient (stressors, neurostimulation) or permanent (lesion) impairment compromises adaptive behavior [42].

Men and women have differences in the dlPFC-amygdala relationship [40], and this may be part of the reason for gender-based emotionality differences (in addition to NR3A differences+).

Guanfacine (which increases dlPFC delay cell firing through α2A-adrenergic agonism) has already been shown to change the dlPFC-amgydala relationship to enhance cognitive control [37], however its upside has been limited by other effects of α2A-adrenergic receptor agonism, though it is still interesting to investigate.

Cognitively Enhancing Effects of GCP-II inhibition

NAAG function is highly correlated with IQ. For people with the gene rs202676, they have Decreased NAAG Levels (due to more GCP-II/FOLH1), and they have lower IQ on average as a result, due to lowered mGluR3 [55]. This shows that mGluR3 is not just relevant for memory and executive function, but also IQ and spatial intelligence.

GCP-II inhibition has consistently been able to show a very significant cognitive enhancement in many healthy models.

In one study on young mice, ZJ43 (a GCP-II inhibitor) increased the long-term (1 day) memory of mice, with a significant enhancement of recognition memory in the test. [56]

Mice treated with ZJ43 at doses of 100 mg/kg and 150 mg/kg spent significantly more time exploring a novel object compared to a familiar object when tested 24 hours after initial exposure. This indicates ZJ43 enhanced memory for the familiar object. This was also replicated in a very similar study [57].

Mice lacking the NAAG-inactivating enzyme glutamate carboxypeptidase II (GCPII knockouts) also showed enhanced novel object recognition, mimicking the effect of the GCP-II inhibitors.

GCP-II inhibitors were also tested on rheusus monkeys, and the study showed working memory enhancement [58], with a greater significance for older monkeys.

It is also very likely that GCP-II inhibition increases visualization capabilities as the primary visual cortex (V1) and dlPFC have opposing functions: V1 processes visual stimuli as they occur, while dlPFC, particularly dlPFC Delay cells, represents visual stimuli in their absence [43]. Knowing GCP-II inhibition enhances delay cell activity, it is very possible that inhibiting GCP-II increases visualization memory.

Secondary Effects of GCP-II inhibition

GCP-II inhibition has many known and potential effects, so instead of typing them out, I ordered them into bullet points:

• Analgesia - GCP-II inhibition has shown to be effective against pain in 15+ studies [16], however moreso for neuropathic pain than other pain modalities. This is most likely due to increased mGluR3 changing the pain perception axis through dlPFC modulation. [13][15]
• Treating Addiction - GCP-II inhibition has been shown to be effective against addiction in many models including cocaine [36], alcohol, morphine, overeating (dlPFC) [35], pornography (dlPFC) [29][30], internet addiction (dlPFC) [31] and others [16]. Another interesting effect of GCP-II inhibition is it reverses ethanol impairment in rodent models. [18]
• Treating Schizophrenia/Psychosis - mGluR3 is known to have common dysfunction in schizophrenic patients [17], and GCP-II inhibitors have been shown to be effective in experimental rodent models of PCP induced motor activation [19].
• Treating ADHD - The dlPFC is a known area of dysfunction in persons with ADHD disorder [20][21] with impairment in both hemispheres [22]. Experimental tDCS on patients improved cognitive control [23], but did not attentuate action cancellation, which may suggest NR2D positive modulation in combination with GCP-II inhibition would a good combination for treating ADHD.
• Treating OCD - The dlPFC is also very relevant in obsessive-compulsive disorder [25][26][27][28]. OCD is more to do with the dlPFC-OFC (orbitofrontalcorex) relationship than ADHD. In one study [24], OCD patients demonstrated reduced functional between the right DLPFC and right orbitofrontal cortex (OFC), and activity in the right OFC had an inhibitory effect on the dlPFC. This may suggest that OCD is potentially rooted in a dlPFC-OFC relationship where the OFC has a higher cognitive control, stopping the dlPFC's inhibitory control and high-level cognitive control. "The OFC is a major inhibitor of the self-control function of the DLPFC in OCD patients in the resting state, while the DLPFC engages top-down control input to the OFC when emotional task stimulation is applied". This suggests a large potential for GCP-II inhibition in treating OCD (and perhaps combined with a NR2B PAM for further BA24/25 modulation).
• Treating Anhedonia - The dlPFC has implications in anhedonia, due to its role in emotional regulation with the NAc and brodmann areas 24/25 [32]. In one study, rTMS (transcranial stimulation) on the left dlPFC in subjects with MDD showed less markers of anhedonia [33].
• Cancer Inhibition - GCP-II is also known as PSMA (prostate-specific membrane antigen) has been been studied with relevance to cancer extensively. PSMA is mostly located within the brain, however in the prostate, PSMA is found in an 8- to 12-fold increase over levels in noncancerous prostate cells [47], so it has become a relevant target for inhibiting cancer. In one study on pancreatic cancer in mice, 2-PMPA (a GCP-II inhibitor) signficantly reduced tumor weight, and when combined with CB839 (glutaminase inhibitor) the combination nearly completely inhibited new growth [46].
• Reversing/Treating Alzheimers - The dlPFC is significantly different in structure and receptor distribution compared to other parts of the brain [43], and because of this, it is more vulnerable to age-based degradation. GCP-II inhibition was shown in one study [44] to reverse cognitive memory deficits in one rodent model of Alzheimer’s disease. Increasing NAAG via GCP-II inhibition also has many other neuroprotective effects. GCP-II inhibition was shown to protect against hypoxia (up to 100%) in one study [49], and also protecting to a lesser extent against NMDA and glutamate injury. In addition, mGluR3 Positive Modulation can induce an increased production of GDNF and TGF-b [50], in a mGluR3-dependent manner.
• Lifespan Enhancement - GCP-II inhibition has been shown to increase lifespan by 10-15% in one study on mice [45], potentially through many different mechanisms (cancer inhibition, neuroprotection, increased will to live (?)), showing a similar efficacy to rapamycin [48].
• Treating COVID-19 "Brain Fog" - COVID increases GCP-II very significantly [51][52][54], more than other common viruses, leading to impaired cognition, poor executive function and motivation (and more) through lowered mGluR3 in the dlPFC [53][54]. This happens in long covid as well, so currently the most effective novel approach in removing long-covid seems to be a combination of GCP-II inhibition, KAT-II inhibition and potentially dlPFC-specific prTMS.

GCP-II Inhibiting Compounds

There have been multiple GCP-II inhibiting compound through the years, but to make a long story short, 2-MPPA was the first (major) candidate, but it was withdrawn from use due to safety concern. It went through a safety trial in humans and at its effective dose it was well tolerated, however due to the thiol group on the structure, it could cause hepatotoxicity.

Due to this, a new small-molecule inhibitor of GCP-II was created, called 2-PMPA. This compound got rid of the thiol group, however it sacrified oral dose efficacy in the process (meaning intranasal or IV is likely the best dose method).

The best candidates for GCP-II inhibition look to be 2-PMPA and ZJ43. Both of them have proven useful in increasing delay cell firing, with 2-PMPA being able to double delay cell firing. Both compounds have excellent safety predictions in Admetlab 2.0, and ZJ43 is an amino acid conjugate.

2-PMPA has worse BBB penetration and used to have offtarget concerns at cytosolic carboxypeptidases (CCPs), but the offtarget concerns ended up being insignificant.

2-PMPA and ZJ43 would likely work best intranasally, at 50-100mg approx human dose. Intranasal dosage of 2-PMPA significantly increases its half life and also brain penetration [59]. After 30mg/kg (a lot) of 2-PMPA dosed intranasally in monkeys, it completely inhibited GCP-II in the cortex.

Comparing the two, 2-PMPA is a smaller compound, so it may be cheaper to produce. On the other hand, ZJ43 is also promising and shows an equal efficacy for cognitive enhancement. These compounds are still experimental and not trialed in humans, however they have shown a great safety profile in the studies we do have.

Here at Penchant, we plan to synth a GCP-II inhibiting compound within the next few months, so that it can be used professionally for analysis and testing.

Final Notes

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