The MET-FLAM Faculty

Aitak FARZI

Scientific Interests:

We demonstrated that high fat diet (HFD) feeding induces distinct alterations of intestinal microbiome and behavioral changes indicative of depression-like behavior as revealed by reduced sociability and anhedonia assessed through sucrose preference. These effects were paralleled by changes in the metabolome of prefrontal cortex and striatum, changing the relative concentrations of molecules involved in energy metabolism (e. g. lactate) and neuronal signaling (e. g. γ-aminobutyric acid) [2]. We further observed that depletion of intestinal bacteria with a combination of non-absorbable antibiotics prevented HFD-induced sucrose preference and self-care reduction, suggesting that signals derived from the intestinal microbiome contribute to HFD-induced behavioral disturbances [3]. Indeed, intestinal microbiota-related metabolites including short-chain fatty acids (SCFAs) were decreased in the antibiotic-treated group and were associated with decreased circulating levels of the adipose tissue hormone leptin. The anhedonic effect of HFD was absent in leptin-deficient ob/ob mice although these animals gained more weight in response to HFD, indicating that intestinal microbiome-dependent changes in leptin levels contribute to HFD-induced anhedonia [3]. We further demonstrated that combining HFD with psychological stress aggravates diet-induced obesity with central insulin resistance of central amygdalar neuropeptide Y neurons mediating the accelerated weight gain [6]. In addition to neuropeptide Y, we described the distinct and region-specific effects of the neuropeptide cocaine- and amphetamine-regulated transcript (CART) in the arcuate nucleus of the hypothalamus and lateral hypothalamic area on food intake and energy homeostasis in response to overexpression of CART and activation of CART neurons [7, 8].

While under obese conditions, antibiotic-induced reduction of the “dysbiotic” gut microbiome attenuates diet-induced depression-like behavior, we described that antibiotic-induced depletion of the gut microbiota leads to cognitive impairment, specifically impaired novel object recognition but not spatial memory [9]. This behavioral change was associated with a disruption of the microbial community in the colon, distinct alterations of the colonic and circulating metabolite profile (lipid species and converted bacteria-derived molecules) and particular changes of neurochemical brain activity (e. g. BDNF and serotonin transporter expression) [9].

Various bacterial metabolites including SCFAs are suggested to exert positive effects on brain function through multiple mechanisms. In addition, the gut microbiota is a rich source of microbe-associated molecular patterns (MAMPs), conserved microbial motifs that are recognized by pattern recognition receptors (PRRs) of the host that are able to induce innate immune responses. We characterized immune and central responses of various MAMPs including the bacterial peptidoglycan fragments heptanoyl-gamma-D-glutamyl-(L)-meso-diaminopimelyl-(D)-alanine (FK-565) and muramyl dipeptide (MDP), which activate nucleotide-binding oligomerization domain-containing protein (Nod) 1 and 2, respectively [10, 11, 12, 13, 14]. We could demonstrate that pharmacological administration of peptidoglycan fragments induces mild peripheral and central immune responses and mild induction of c-Fos expression, an indicator of neuronal activation, in distinct brain areas including the central amygdala. On the behavioral level, Nod1 activation induced behavioral changes indicative of sickness behavior, while Nod2 activation by MDP did not induce a sickness response. Combining these peptidoglycan fragments with the outer membrane component of Gram-negative bacteria, lipopolysaccharide, however, synergistically amplified the immune, sickness and brain responses to peripheral immune stimulation [10].

Proposed Dissertation Topic:

Background: Obesity is a risk factor for cognitive impairment and mood disorders such as major depression. Western-style dietary patterns contribute to obesity-related complications through changes in gut microbiome composition, resulting in inflammatory and endocrine alterations that ultimately affect brain function and behavior. Specifically, challenging mice with a high-fat diet (HFD) leads to central inflammatory processes and insulin resistance that both contribute to synapto-dendritic and behavioral abnormalities. Interestingly, bacterial cell-wall-derived muramyl dipeptide (MDP) blunted HFD-induced adipose tissue inflammation and glucose intolerance by signaling through Nod2 and the transcription factor interferon regulatory factor 4 (IRF4), whereas Nod1 activation exerted opposite effects. However, the central effects of these peptidoglycan derivates in the context of HFD-induced obesity are unknown. Potential central effects of peptidoglycans in the context of obesity are, however, of great importance, as they cross the blood brain barrier and affect central immune responses.

Hypothesis and objectives: We hypothesize that Nod1 and Nod2 signaling plays an important role in HFD-induced cognitive impairment and depression by modulating immune activation and insulin resistance in the brain. Our aims are to delineate the effects of bacterial peptidoglycans, including the Nod1 agonist FK565 and the Nod2 agonist MDP, on HFD-induced changes in central immune parameters, central insulin resistance, brain function, and behavior. Furthermore, we aim to assess differences in downstream signaling in response to Nod1 and Nod2 activation.

Methods and approaches: The PhD candidate will perform in vivo mouse studies that involve the analysis of Nod1 and Nod2 signaling on HFD-induced behavioral disturbances and associated changes in brain function. To this end, test batteries of affective behavior will be used to study social interaction, depression-like behavior, anxiety, learning, and memory (1st and 2nd year). In addition, the student will perform insulin sensitivity and glucose tolerance tests and determine central insulin signaling by quantification of insulin receptor expression and insulin receptor substrate 1 phosphorylation in various brain areas. Downstream signaling in response to Nod1 and Nod2 activation will be assessed by IRF4 expression and NFκB activity assessed by TransAM^® NFκB Activation Assay. Central immune activation will be analyzed by quantifying pro-inflammatory cytokines (TNFα, IL-1β, IL-6, and IL-10) (3rd year), while microglial signatures will be studied by determining microglia markers including Iba-1 and CD68 by immunohistochemistry and flow cytometry. Brain synapse density and other ultrastructural features will be evaluated by electron microscopy (4th year).

Pitfalls and alternative approaches: The methods and techniques used in this project are established and part of routine lab work, so we do not expect to encounter major technical problems. Previously, we administered the Nod agonists via intraperitoneal administration. However, recent research has demonstrated that peptidoglycan trafficking is activated by the gut microbiota, and oral administration surprisingly leads to higher central levels of peptidoglycans [15]. Consequently, we will administer the Nod agonists under study via the oral route. Importantly, exogenously administered peptidoglycan results in high concentrations of peptidoglycan in brain tissue, highlighting the significance of central peptidoglycan signaling. If, contrary to expectation, we fail to observe central effects of the Nod agonists under study, we will expand our analysis to other peptidoglycan-responsive pattern-recognition receptors known to have central effects, such as PGN-recognition protein 2 [16].

Involved Faculty members: Aitak Farzi (PI), Julia Kargl (flow cytometry), Dagmar Kratky (metabolic phenotyping), Stefano Angiari (Nod signaling).

International Collaborations: Kenny (Chi Kin) Ip (Garvan Institute of Medical Research, Sydney, AUS).

Facilities: Our team currently consists of one PhD candidate, one master student, personnel trained in mouse maintenance and handling, and one technicians. Our laboratory is located on the newly opened basic research campus of the university, just opposite the university hospital. Our division provides the required laboratory and office space, secretarial assistance, and basic laboratory facilities, such as laboratories for behavioral experiments, flow cytometry, immunofluorescence microscopy and more. Specific equipment for mouse behavioral testing are also available in our lab. LabMaster Metabolic Cage system for metabolic phenotyping, electron microscopy and metabolomics are also available on the campus through collaboration partners.

Preparatory Findings:

Figure 1: Eight week feeding with high fat diet (48 % Kj fat) induces anhedonia and modulates central immune responses.
(A) Sucrose preference in response to 8 weeks of high fat diet (HFD) feeding (Student’s t-test). (B) Increased expression of inflammatory cytokines in response to HFD in the hypothalamus assessed by qPCR (two-way ANOVA with main effect of HFD). (C) RNA sequencing data indicating increased expression of Nod2 in response to HFD. Data are shown as mean + SEM, n = 6–10, * p < 0.05.

1. Farzi A, Fröhlich EE, Holzer P: Gut Microbiota and the Neuroendocrine System. Neurotherapeutics, 2018; 15(1):​5–22.
   
2. Hassan AM, Mancano G, Kashofer K, Fröhlich EE, Matak A, Mayerhofer R, Reichmann F, Olivares M, Neyrinck AM, Delzenne NM, Claus SP, Holzer P: High-fat diet induces depression-like behaviour in mice associated with changes in microbiome, neuropeptide Y, and brain metabolome. Nutr Neurosci, 2019; 22(12):​877–893.
   
3. Hassan AM, Mancano G, Kashofer K, Liebisch G, Farzi A, Zenz G, Claus SP, Holzer P: Anhedonia induced by high-fat diet in mice depends on gut microbiota and leptin. Nutr Neurosci, 2022; 25(2):​299–312.
   
4. Farzi A, Ip CK, Reed F, Enriquez R, Zenz G, Durdevic M, Zhang L, Holzer P, Herzog H: Lack of peptide YY signaling in mice disturbs gut microbiome composition in response to high-fat diet. FASEB J, 2021; 35(4):​e21435.
   
5. Farzi A, Hassan AM, Zenz G, Holzer P: Diabesity and mood disorders: Multiple links through the microbiota-gut-brain axis. Mol Aspects Med, 2019; 66:​80–93.
   
6. Ip CK, Zhang L, Farzi A, Qi Y, Clarke I, Reed F, Shi YC, Enriquez R, Dayas C, Graham B, Begg D, Brüning JC, Lee NJ, Hernandez-Sanchez D, Gopalasingam G, Koller J, Tasan R, Sperk G, Herzog H: Amygdala NPY Circuits Promote the Development of Accelerated Obesity under Chronic Stress Conditions. Cell Metab, 2019; 30(1):​111–128.e6.
   
7. Lau J, Farzi A, Qi Y, Heilbronn R, Mietzsch M, Shi YC, Herzog H: CART neurons in the arcuate nucleus and lateral hypothalamic area exert differential controls on energy homeostasis. Mol Metab, 2018; 7:​102–118.
   
8. Farzi A, Lau J, Ip CK, Qi Y, Shi YC, Zhang L, Tasan R, Sperk G, Herzog H: Arcuate nucleus and lateral hypothalamic CART neurons in the mouse brain exert opposing effects on energy expenditure. ELife, 2018; 7:e36494
   
9. Fröhlich EE, Farzi A, Mayerhofer R, Reichmann F, Jačan A, Wagner B, Zinser E, Bordag N, Magnes C, Fröhlich E, Kashofer K, Gorkiewicz G, Holzer P: Cognitive impairment by antibiotic-induced gut dysbiosis: Analysis of gut microbiota-brain communication. Brain Behav Immun, 2016; 56:​140–155.
   
10. Farzi A, Reichmann F, Meinitzer A, Mayerhofer R, Jain P, Hassan AM, Fröhlich EE, Wagner K, Painsipp E, Rinner B, Holzer P: Synergistic effects of NOD1 or NOD2 and TLR4 activation on mouse sickness behavior in relation to immune and brain activity markers. Brain Behav Immun, 2015; 44:​106–120.
    
11. Mayerhofer R, Fröhlich EE, Reichmann F, Farzi A, Kogelnik N, Fröhlich E, Sattler W, Holzer P: Diverse action of lipoteichoic acid and lipopolysaccharide on neuroinflammation, blood-brain barrier disruption, and anxiety in mice. Brain Behav Immun, 2017; 60:​174–187.
    
12. Painsipp E, Köfer MJ, Farzi A, Dischinger US, Sinner F, Herzog H, Holzer P: Neuropeptide Y and peptide YY protect from weight loss caused by Bacille Calmette-Guérin in mice. Br J Pharmacol, 2013; 170(5):​1014–1026.
    
13. Zenz G, Farzi A, Fröhlich EE, Reichmann F, Holzer P: Intranasal Neuropeptide Y Blunts Lipopolysaccharide-Evoked Sickness Behavior but Not the Immune Response in Mice. Neurotherapeutics, 2019; 16(4):​1335–1349.
    
14. Zenz G, Jacan A, Reichmann F, Farzi A, Holzer P: Intermittent Fasting Exacerbates the Acute Immune and Behavioral Sickness Response to the Viral Mimic Poly(I:C) in Mice. Front Neurosci, 2019; 13:​359.
    
15. Wheeler R, Bastos PAD, Disson O, Rifflet A, Gabanyi I, Spielbauer J, Bérard M, Lecuit M, Boneca IG: The bacterial peptidoglycan-sensing molecule Pglyrp2 modulates brain development and behavior. Proc Natl Acad Sci USA, 2023; 120(4):​e2209936120.
    
16. Arentsen T, Qian Y, Gkotzis S, Femenia T, Wang T, Udekwu K, Forssberg H, Diaz Heijtz R: The bacterial peptidoglycan-sensing molecule Pglyrp2 modulates brain development and behavior. Mol Psychiatry, 2017; 22(2):​257–266.