**5. Microbiome**

In conclusion, maturation of the autonomic nervous system may be delayed in preterm and IUGR animals. Furthermore, delayed development of the GIT in preterm and IUGR animals, including longer gut permeability, facilitates the toxic effect of external factors including bacterial translocation. Furthermore, the immature gut seemingly fails to stimulate the development of the vagus nerve. Importantly, there is some evidence pointing to altered gut permeability (leaky gut) in autism and possibly genetic predisposition to abnormalities in tight

**Figure 1. Packetts of apoptotic cells in the epithelium of neonatal piglets.** Massive apoptosis is evidenced by scan‐ ning electron microscope (SEM) image by shortened microvilli, several yet unzipped spaces between cells are present.

(SEM images generously supplied by dr. Tomasz Skrzypek, Catholic University of Lublin, Poland)

shortened microvilli, several yet unzipped spaces between cells are present.

Fig. 1. **Figure 1. Packetts of apoptotic cells in the epithelium of neonatal piglets.** Massive apoptosis is evidenced by scanning electron microscope (SEM) image by

(SEM images generously supplied by dr. Tomasz Skrzypek, Catholic University of

**4. Determinants of individual sensitivity of brain-gut axis and gut**

**microbiome to environmental toxins; intrinsic and extrinsic components**

Studies of the human microbiome revealed that even healthy individuals differ remarkably in the microbes of the gut. The gut microbiome is regulated by both extrinsic and intrinsic factors.

junctions in ASD (White, 2003; de Magistris et al, 2010).

Lublin, Poland)

Fig. 1.

66 Recent Advances in Autism Spectrum Disorders - Volume I

The human GIT harbors a large number (1000 to 1150) of bacterial species and is involved in maintaining homeostasis and well-being. Functions of this microbiome include the regulation of the mucosal immune system, GIT motility, epithelial barrier regulation, gut secretion, digestion and metabolism (Grenham et al., 2011). One of the main functions of gut microbes is to extract nutrients from otherwise indigestible fibers (Tremaroli and Backhed, 2012). The microbiome, absent at birth, is gradually colonized by facultative bacteria and anaerobic bacteria (Grenham et al., 2011).

Several lines of evidence point to both brain-gut axis and gut microbiome abnormalities in autism which are summarized in Fig 3. Children with ASD frequently present a variety of gastrointestinal (GI) symptoms, although some claim that the data supporting increased GI symptomology in autistic children not to be rigorous enough (Erickson et al., 2005). The socalled "bacterial theory" of autism proposes the GIT symptoms are associated with changes in microbial composition and that these changes could be involved in the pathogenesis or progression of several childhood diseases including autism (Somma et al., 2010).

**Figure 2. The effect of LPS exposure on cerebellar gene expression.** Gene expression **Figure 2. The effect of LPS exposure on cerebellar gene expression.** Gene expression was measured by quantita‐ tive RT-PCR in cerebellar tissue of rat pups exposed perinatally to LPS (200μg/kg BW) and was normalized to cyclophi‐ lin A. Panel A: males, Panel B: females. Data are presented as relative gene expression (mean±S.E.M.; \*, p< 0.05; +, p< 0.1; Xu et al., submitted).

was measured by quantitative RT-PCR in

It has been suggested that an abnormal gut microbiome in some ASD children may be due to certain antimicrobial drugs that play a key role in modifying the intestinal bacterial flora and selecting potentially harmful bacteria normally kept at bay by the innate intestinal flora. And so, both Clostridia (Finegold, 2011b) and Desulfovibrio (Finegold, 2011a) have been implicated in autistic pathology. Clostridia form spores and the spores could likely survive antibiotic treatment and subsequently flourish. Desulfovibrio is an anaerobic bacillus that does not produce spores and is resistant to some antibiotics such as cephalosporins used in treatment of common childhood diseases such as ear infections (Finegold, 2011a). An increase in Bacteroides, a decrease in Firmicutes with an overall increase in biodiversity has been observed in IBD, celiac disease and autism (Iebba et al., 2011). An increase in *Clostridium histolyticum*, a recognized toxin producer with systemic effects, has been observed in fecal samples of ASD children (Parracho et al., 2005). A strong correlation of gastrointestinal symptoms with autism, and a decrease in Bifidobacteria and increase in Lactobacilli, was observed in fecal samples of ASD children (Adams et al., 2011). An association between high levels of intestinal, mucoepi‐ thelial-associated Sutterella species and GI disturbances has been detected in intestinal biopsy samples in children with autism (Williams et al., 2012). This latter study may provide the most accurate picture of the gut microbiome as the data were derived directly from the gut. cerebellar tissue of rat pups exposed perinatally to LPS (200µg/kg BW) and was normalized to cyclophilin A. Panel A: males, Panel B: females. Data are presented as a relative gene expression (mean±S.E.M.; \*, p< 0.05; +, p< 0.1; Xu et al., submitted).

Our most recent studies suggest altered expression of ghrelin, the activating enzyme (ghrelin O-acyltransferase, GOAT) and the receptor in several brain areas of autistic children (Sajdel-Sulkowska, unpublished observation). A decrease in ghrelin mRNA has been also observed in the temporal gyrus of Alzheimer patients (Gahete et al., 2010) suggesting ghrelin may contribute to the severity of AD pathology. Since we have measured the levels of ghrelin mRNA, it can be assumed that the changes observed were due to the altered levels of brain

**Figure 3.** Altered gut microbiome and the brain gut axis in autism. S-R-CTR, social reward center; F-R CTR, food reward

reward center; F-R CTR, food reward center.

**Figure 3. Altered gut microbiome and the brain gut axis in autism.** S-R-CTR, social

F-R CTR

> S-R CTR

Gut Microbiome and Brain-Gut Axis in Autism — Aberrant Development of Gut-Brain Communication…

STRIATUM

 **AUTISM** \*Altered social and feeding behaviors

motility

\*Altered function(s) of social and food-reward center(s)?? \*Gastrointestinal problems

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69

\*Increased gut permeability " leaky-gut"; altered gut

\*Increased levels of cytokines \*Altered levels of brain neurotrophis \*Increased biodiversity of gutmicrobiome; dysbiosis

The majority of circulating ghrelin is synthesized by gastric mucosa X/A-like cells in response to negative energy status. These cells are not typical endocrine cells since the oxyntic mucosa cells produce HCl in the stomach lumen and ghrelin as a hormone. Ghrelin is the most potent orexigenic peptide, and plays an important role in glucose metabolism and also in GIT cytoprotection. In addition to its ability to stimulate appetite, ghrelin stimulates the release of growth hormone release via the growth secretagogue, GHS-R1a receptor. Ghrelin O-acyl‐ transferase, GOAT, is the enzyme that activates ghrelin. The ghrelin/GHS-R/GOAT system may play an important role in metabolic disorders in children (Lim and Korbonits, 2012). In addition to the ghrelin of GIT origin, and the hypothalamus being the main source of brain ghrelin, ghrelin has been detected in the midbrain, hindbrain, hippocampus, spinal cord and

ghrelin.

center.

Fig.3.

 **HEALTH** \*Normal range of social and feeding behaviors \*Functional social- and foodreward center(s) \*Normal gastrointestinal functions

motility

\*Normal gut permeability and gut

\*Normal level of cytokines \*Age-appropriate level of brain neurotrophins \*Balanced gut microbiome

A response to oral treatment with vancomycin, not absorbed from the GI tract, in autism suggests the importance of gut flora in a disease (Finegold, 2011a). Evidence suggests that ASD may be associated with altered innate immune response; thus children with GI problems may reflect inflammation as a reaction to an endotoxin produced by gut bacteria (Jyonou‐ chi et al, 2002).

Fig.3.

**Figure 3. Altered gut microbiome and the brain gut axis in autism.** S-R-CTR, social reward center; F-R CTR, food reward center. **Figure 3.** Altered gut microbiome and the brain gut axis in autism. S-R-CTR, social reward center; F-R CTR, food reward center.

It has been suggested that an abnormal gut microbiome in some ASD children may be due to certain antimicrobial drugs that play a key role in modifying the intestinal bacterial flora and selecting potentially harmful bacteria normally kept at bay by the innate intestinal flora. And so, both Clostridia (Finegold, 2011b) and Desulfovibrio (Finegold, 2011a) have been implicated in autistic pathology. Clostridia form spores and the spores could likely survive antibiotic treatment and subsequently flourish. Desulfovibrio is an anaerobic bacillus that does not produce spores and is resistant to some antibiotics such as cephalosporins used in treatment of common childhood diseases such as ear infections (Finegold, 2011a). An increase in Bacteroides, a decrease in Firmicutes with an overall increase in biodiversity has been observed in IBD, celiac disease and autism (Iebba et al., 2011). An increase in *Clostridium histolyticum*, a recognized toxin producer with systemic effects, has been observed in fecal samples of ASD children (Parracho et al., 2005). A strong correlation of gastrointestinal symptoms with autism, and a decrease in Bifidobacteria and increase in Lactobacilli, was observed in fecal samples of ASD children (Adams et al., 2011). An association between high levels of intestinal, mucoepi‐ thelial-associated Sutterella species and GI disturbances has been detected in intestinal biopsy samples in children with autism (Williams et al., 2012). This latter study may provide the most accurate picture of the gut microbiome as the data were derived directly from the gut.

**Figure 2. The effect of LPS exposure on cerebellar gene expression.** Gene expression was measured by quantita‐ tive RT-PCR in cerebellar tissue of rat pups exposed perinatally to LPS (200μg/kg BW) and was normalized to cyclophi‐ lin A. Panel A: males, Panel B: females. Data are presented as relative gene expression (mean±S.E.M.; \*, p< 0.05; +, p<

**Figure 2. The effect of LPS exposure on cerebellar gene expression.** Gene expression was measured by quantitative RT-PCR in cerebellar tissue of rat pups exposed perinatally to LPS (200µg/kg BW) and was normalized to cyclophilin A. Panel A: males, Panel B: females. Data are presented as a relative gene expression (mean±S.E.M.; \*, p< 0.05; +, p< 0.1; Xu et al., submitted).

SWAP Odf4 DIO2 Cirbp TTR Pcp2 DIO3 BDNF RELN FoxP4

SWAP Odf4 DIO2 Cirbp TTR Pcp2 DIO3 BDNF RELN FoxP4

\* <sup>+</sup> \*

Fig. 2.

68 Recent Advances in Autism Spectrum Disorders - Volume I

0 0.5 1 1.5 2 2.5 3 3.5 A. MALES

+

B. FEMALES

0 0.5 1 1.5 2 2.5 3 3.5

RELATIVE GENE EXPRESSION

RELATIVE GENE EXPRESSION

A response to oral treatment with vancomycin, not absorbed from the GI tract, in autism suggests the importance of gut flora in a disease (Finegold, 2011a). Evidence suggests that ASD may be associated with altered innate immune response; thus children with GI problems may reflect inflammation as a reaction to an endotoxin produced by gut bacteria (Jyonou‐

chi et al, 2002).

0.1; Xu et al., submitted).

Our most recent studies suggest altered expression of ghrelin, the activating enzyme (ghrelin O-acyltransferase, GOAT) and the receptor in several brain areas of autistic children (Sajdel-Sulkowska, unpublished observation). A decrease in ghrelin mRNA has been also observed in the temporal gyrus of Alzheimer patients (Gahete et al., 2010) suggesting ghrelin may contribute to the severity of AD pathology. Since we have measured the levels of ghrelin mRNA, it can be assumed that the changes observed were due to the altered levels of brain ghrelin.

The majority of circulating ghrelin is synthesized by gastric mucosa X/A-like cells in response to negative energy status. These cells are not typical endocrine cells since the oxyntic mucosa cells produce HCl in the stomach lumen and ghrelin as a hormone. Ghrelin is the most potent orexigenic peptide, and plays an important role in glucose metabolism and also in GIT cytoprotection. In addition to its ability to stimulate appetite, ghrelin stimulates the release of growth hormone release via the growth secretagogue, GHS-R1a receptor. Ghrelin O-acyl‐ transferase, GOAT, is the enzyme that activates ghrelin. The ghrelin/GHS-R/GOAT system may play an important role in metabolic disorders in children (Lim and Korbonits, 2012). In addition to the ghrelin of GIT origin, and the hypothalamus being the main source of brain ghrelin, ghrelin has been detected in the midbrain, hindbrain, hippocampus, spinal cord and

several organs outside the brain. While the systemic endogenous ghrelin exerts a tonic stimulating effect on hypothalamic CRH (Rucinski et al., 2012), its function in the brain includes the modulation of membrane excitability, control of neurotransmitter release, neuronal gene expression, and neuronal survival and proliferation (Ferrini et al., 2009).

healthy volunteers (Messaoudi et al., 2011) provided encouraging results for further studies exploring the concept of microbial targeting of the GIT under pathological conditions includ‐ ing autism. Individually tailored probiotic formulations, enriched in specific strains of gut bacteria, could one day be used in treatments of ASD even as an adjuvant to other treatments.

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**7. Possible connection of gut microbiome and behavior; microbiome and**

The intestinal microbiome participates in the development of the HPA axis (Sudo et al., 2004) and is critical to the development of appropriate stress response later in life, which occurs during a narrow, critical developmental window. This process involves both the regulation of the levels of brain derived neurotrophic factor (BDNF) and NMDA receptors (Sudo et al., 2004). The microbiome also plays an important role in anxiety-like behavior (Messaoudi et al., 2011), depressive behaviors (Neufield et al., 2011; Messaoudi et al., 2011), but the effects are diminished in vagatomized animals, suggesting either the direct communication between the bacteria and the brain (Bravo et al., 2011) or through the brain-gut axis. The latter possibility is an indirect action of bacteria on an afferent vagal pathway via gut immune, endocrine and

Animal studies have also shown that stress can change the composition of the microbiome, where the changes are associated with increased vulnerability to inflammatory stimuli in the GIT (Gareau et al., 2006); here the microbiome plays an important role in memory dysfunction (Gareau et al., 2011). Stress is known to inhibit gut contraction, one of the crucial defense strategies against bacterial colonization of gut mucosa. Early psychological trauma of maternal separation resulted in persistent mucosal barrier dysfunction in neonatal rats, including host defense to luminal bacteria, by mechanisms involving peripheral CRH receptors (Gareau et

Oral antibiotics disrupt the microbiome and favor environment for opportunistic bacteria. *Clostridium tetani*, an anaerobic bacillus produces a potent neurotoxin, tetanus neurotoxin (TeNT) that is transported by the vagus nerve from the GI to the CNS. In the brain TeNT disrupts the release of neurotransmitters by the proteolytic cleavage of synaptobrevin, a synaptic vesicle membrane protein. This inhibition may be related to a variety of behavioral deficits characteristic of autism. Some children with autism treated with anti-clostridia

antibiotics have shown a reduction in stereotyped behavior (Bolte, 1998).

**microbiome regulation of the reward loop in autism?**

**8. The role of the reward system in gut-brain communication, the**

**interaction between food-reward and social-reward systems; altered gut-**

Autism is characterized by both severe deficits in social interaction and communication and significant eating difficulties with a highly restricted range of food choices (Williams et al.,

**behavioral abnormalities in ASD**

enteric nervous system (ENS) controlling mechanisms.

al., 2006).

It has been reported that ghrelin of GIT origin interacts with bacterial toxins (Tiaka et al., 2011) and exerts a protective role in experimental colitis; is it possible that the ghrelin of brain origin plays a protective role as well? If so, changes in the level of brain-derived ghrelin could be detrimental to the developing brain.
