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IS362, an FMO3 inhibitor, prevent TMAO-mediated synaptic deficits in models of Alzheimer’s disease and Diabetes type 2

The current project seeks to better understand the signaling mechanisms of the gut-brain signaling axis on the development of impaired synaptic plasticity. The aspect that diabetes and AD share many similarities has garnered interest for understanding the impact of metabolites from western diets on alteration of behavior, synaptic plasticity and progression of AD. To this, gut dysbiosis has been observed in diabetics and AD patients, resulting in elevated trimethylamine. The liver enzyme Flavine Mono-Oxygenase 3 (FMO3) liver enzyme increases to supra-physiological levels resulting in hepatic insulin resistance and elevated circulating blood glucose levels as well as an increase in trimethyl amine (TMA) metabolism to an oxide (TMAO). TMAO has been observed to induce inflammation, atherosclerosis, and cardiac ischemic injury. Our published findings involving diabetic rodents and 3xTgAD mice reported that FMO3 is induced to supra-physiological levels with increased blood glucose levels and impaired brain insulin signaling. Reports on TMAO levels in patients have observed mild cognitive impaired patients display 3-fold higher levels of TMAO in cerebrospinal fluid (CSF) and patients with full Alzheimer’s disease demonstrate 4-5 fold higher levels of TMAO in CSF, when compared to age and sex matched healthy individuals. Our published work demonstrates that TMAO induces deficits in basal synaptic transmission as well as alterations in long term potentiation. To explain these findings we observed reduced glutamatergic receptor expression in brain slices exposed to TMAO which was due to an increase in the ER stress marker in PERK signaling pathway associated with ER stress.

In response to stress, neurons can alter their molecular and physiological characteristics. Synaptic plasticity is such a response by the neurons to change the strengthening or weakening of synaptic connections between neurons [39]. These activity-dependent, changes in synaptic strength are triggered by de novo protein synthesis [40]). These findings indicate that protein synthesis can be triggered locally at activated synapses and is required for persistent, activity-dependent forms of synaptic plasticity, which in turn is thought to be essential for memory formation. Notably, in multiple species ATF4 and its homologs act as repressors of cAMP-responsive element binding protein (CREB)-mediated gene expression, which is known to be required for long-lasting changes in synaptic plasticity and long term memory. Our findings are consistent with this concept that we observed increased levels of ATF4 and reduced CREB levels. Further that ATF4 also regulates CHOP which results in enhancing inflammation (Figure 1). In the current study we observed reduced LTP in both 3xTg-AD and db/db mice. In addition, both models also displayed reduced AMPAR and NMDAR subunits. Further, we are the first to report that TMAO alters LTP, as demonstrated in isolated healthy brain slices exposed to TMAO. To help explain these findings reduced levels of AMPAR and NMDAR subunits may be responsible for the impairments in LTP induction and preservation. The reduction in glutamatergic receptor subunits including GluA1 and GluN2A are related to a reduction in LTP and basal synaptic transmission. An increase in presynaptic release induced by TMAO is probably endoplasmic reticulum oxidoreductin-1α (ero1α) dependent, which might help explain the increased basal synaptic transmission. However, vGLUT is responsible for the uptake of glutamate into synaptic vesicles at the presynaptic terminals and thus is related to the number and fill state of presynaptic vesicles and release probability in hippocampal neurons. In the current study, the increase in VGLUT1 expression suggest an increase in the availability of presynaptic vesicles and the presynaptic release probability in TMAO incubated slices.

Based upon our published findings, we suggest that TMAO influences the hippocampal synaptic transmission by altering  glutamatergic signaling pathway (Figure 1). Our results suggest that the elevated FMO3 in the liver induces an increase in TMAO which induces ER stress in the brain. The protein misfolding associated with elevated TMAO results in impaired synaptic transmission and LTP ( Previous studies have demonstrated that TMAO induces inflammation and oxidative stress. Similarly, our results indicate that TMAO induces oxidative stress by increasing ROS, hydrogen peroxide levels, lipid peroxidation and nitrite content in hippocampal slices. Furthermore, oxidative stress is strongly associated with cognitive impairment, leading to reduced life quality and expectancy. Hence, the results from our study indicate a strong association between TMAO and cognitive deficits. Further work will explore the impact of TMAO on spine density via Cofilin activation and actin dynamics. We have recently developed inhibitors of FMO3 to target reduction of TMAO reduction resulting in attenuating the cognitive deficits seen in AD and other diseases with cognitive impairment. We have developed a library of FMO3 inhibitors that will help reduce bacterial-derived TMAO as well as hepatic-induced TMAO production. Our compounds have been designed to have high and poor gut permeability thus impacting the gut microbiome and or liver enzymes. The current study will be applied to an R01 for a better understanding of the impact of metabolites from the gut to the brain in diabetic and Alzheimer’s disease patients. Please see current publication

T Activation of PERK by TMAO leads to phosphorylation of eIF2α which can directly inhibit general protein synthesis (mRNA translation) or activate ATF4.

Figure 1 T Activation of PERK by TMAO leads to phosphorylation of eIF2α which can directly inhibit general protein synthesis (mRNA translation) or activate ATF4. ATF4 negatively regulates synaptic plasticity and memory by acting as a repressor of CRE-dependent transcription (CREB). Alternatively, ATF4 translocate to the nucleus and increases the expression of CHOP which promotes reactive oxygen species formation, inflammation, and apoptosis.

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