Adolescence and Aging: Impact of Adolescence Inflammatory Stress and Microbiota Alterations on Brain Development, Aging, and Neurodegeneration

Abstract Puberty/adolescence is a critical phase during neurodevelopment with numerous structural, neurochemical, and molecular changes occurring in response to genetic and environmental signals. A consequence of this major neuronal reorganizing and remodeling is a heightened level of vulnerability to stressors and immune challenges. The gut microbiota is a fundamental modulator of stress and immune responses and has been found to play a role in mental health conditions and neurodegenerative disorders. Environmental insults (stress, infection, neuroinflammation, and use of antibiotics) during adolescence can result in dysbiosis subsidizing the development of brain disorders later in life. Also, pubertal neuroinflammatory insults can alter neurodevelopment, impact brain functioning in an enduring manner, and contribute to neurological disorders related to brain aging, such as Alzheimer’s disease, Parkinson’s disease, and depression. Exposure to probiotics during puberty can mitigate inflammation, reverse dysbiosis, and decrease vulnerabilities to brain disorders later in life. The goal of this review is to reveal the consequences of pubertal exposure to stress and immune challenges on the gut microbiota, immune reactivity within the brain, and the risk or resilience to stress-induced mental illnesses and neurodegenerative disorders. We propose that the consumption of probiotics during adolescence contribute to the prevention of brain pathologies in adulthood.

The gut microbiota is composed of nearly 100 trillion bacteria that are essential for health. These commensal microorganisms are important for the development of the immune system, protection of the host against pathogens, and metabolism of dietary nutrients and drugs (1). The gut microbiota is dynamic and changes throughout the life span. Maturation of the gut microbiota occurs in parallel with neurodevelopment and they both have similar critical developmental periods. In this review, we aim to reveal the impact of the gut microbiota on the pubertal brain. We highlight the enduring consequences of pubertal exposure to stress and immune challenges (bacterial, viral and parasitic infections, inflammatory conditions) on the gut microbiota, neuroinflammation, and stress-induced neurodegenerative disorders as well as suggest new directions for the development of effective therapeutic interventions.

Puberty/Adolescence and hypothalamic-pituitaryadrenal axis
In humans and animals, puberty/adolescence is a critical period of development during which secondary sexual characteristics and reproductive capability are attained (2). This period is accompanied by numerous hormonal, behavioral, and social changes that impact behavior later in life (2,3). One of the critical systems that develop and mature during puberty/adolescence is the hypothalamic-pituitary-adrenal (HPA) axis (4). In rats, basal levels of stress hormones remain constant throughout pubertal/adolescent development (5). While exposure to physical and psychological stressors increases corticosterone concentration in pubertal and adult rats, the increase in corticosterone concentration is significantly greater in pubertal rats compared with adults (6). Moreover, upon the termination of an acute stressor, the hormonal stress response (adrenocorticotropic hormone [ACTH] and corticosterone) takes longer to return to baseline in prepubertal male and female rats (28 days of age) compared with adults (77 days of age) (4) The heightened stress response in pubertal rats could be explained by the fact that the negative feedback process in the HPA axis is not fully developed and could slow the recovery of the HPA axis following stress exposure. Conversely, following chronic stress, peri-pubertal male rats (28 days of age) display greater HPA axis reactivity and faster return to baseline compared with adults (77 days of age) (6,7). This age difference in HPA axis activity following acute and chronic stress and different types of stressors is associated with greater production of corticotropin-releasing hormone (CRH) in the paraventricular nucleus (PVN) in peri-pubertal compared with adult rodents. Therefore, vulnerability to stress increases during puberty/ adolescence, and individuals are affected differently by different types of stressors during this critical period of development.

Neurodevelopment During Puberty and Adolescence
During puberty/adolescence, the brain undergoes significant reorganizing and remodeling leading to neurogenesis, synaptogenesis, and axonal and dendritic growth. This structural reshaping of neural circuits is important for brain function and behavior later in life (8). The first major change during adolescence is neuronal reorganization which is associated with the refinement of social and cognitive abilities, reward sensitivity, and stress responsivity (8,9). Multiple brain areas undergo morphological changes. For example, the frontal cortex, which is implicated in executive function, cognition, and memory, undergoes changes in synaptic connectivity and axonal myelination (10,11). The synapse number, as well, is modified during puberty and adult synapse quantity is attained by mid-adolescence (10,12). Synapse refinement continues throughout adolescence and into adulthood; it involves synaptic remodeling without any important loss of neurons with the establishment and consolidation of newly formed neural circuits. However, exposure to certain environmental factors during puberty can disrupt this neurodevelopmental process and lead to enduring, or even permanent, changes in brain function and behavior (13,14).
The second major change during adolescence is related to the process of myelination. Myelin is produced by glial cells and forms an insulating sheet around axons, leading to an increased speed of neural communication. Although in humans, axons are fully myelinated in the sensory and motor cortices during the first few years of life, the process of myelination continues in the frontal cortex throughout adolescence (15). In rats, myelin basic protein emerges 6-8 days after birth, peaks at about 15 days postnatal, and decreases thereafter (16). Thus, an increase in the speed of neural communication is observed in the frontal cortex during adolescence when compared with early childhood (17,18). In addition, gray matter volume increases in the frontal and parietal lobes throughout childhood and puberty, and then it decreases progressively (19,20). Brain areas subject to myelination show an increase in white matter volume (21). It is important to note that pruning and myelination modifications are directly associated with changes in gray and white matter volumes in addition to alterations in synaptic connectivity (19,22). These structural changes are related to changes in brain function during adolescence. For example, adolescents show some performance deficiencies in cognitive performance when task loads increase. This performance deficit is related to reduced frontal brain activity and a greater involvement of emotional and motivational subcortical regions (23). Teens may display altered striatal responses to rewards. For example, there is an increase in reward-seeking behavior during mid-adolescence, which then decreases progressively into adulthood (24,25). In addition, social interactions increase during adolescence. Rodents and nonhuman primates show more social interactions as well as greater novelty-seeking behavior during adolescence (26,27). These behavioral changes are linked to dendritic pruning in the amygdala, nucleus accumbens, and prefrontal cortex and continued to increase in fiber density between the amygdala and prefrontal cortex into early adulthood (22,26,28,29). A consequence of this important neuronal rewiring during adolescence is high vulnerability to certain environmental factors, such as stress, drugs, infections, dietary deficiencies, inflammation, and more (26).

Enduring Effects of Pubertal Immune Challenge on Brain Function
Pubertal Immune Signals and Maturation of Microglia The immune system and its cytokine signaling molecules play a key role in neurodevelopment and contribute to synapse formation, refinement, neurogenesis, neuronal differentiation, and response to injury (30,31). For example, both IL-1β and TNFα can inhibit excitatory synaptic transmission (32,33) and are implicated in synapse refinement and plasticity in the cerebellum, hippocampus, and cortex (30). Microglia, immune cells residing in the brain, play a major role in the elimination of certain synapses and in the maintenance of others (34). During synaptic pruning, frequently used synapses are strengthened, whereas scarcely used ones are eliminated (35). In sensory brain regions, synaptic density attains adult levels around adolescence (35,36). The prefrontal cortex, as well, undergoes synapse proliferation during childhood and puberty, followed by stability in synaptic density, and a subsequent postpubertal elimination and reorganization of synaptic connections (10,37,38). During adolescence, activated microglia have ameboid shape and this status is associated with elevated levels of cytokines. The degree of microglial colonization is affected by certain environmental factors (ie, stressors) (39). In response to a stressor, there is activation of the hypothalamic-pituitary-adrenal axis and the sympathetic-adrenal-medullary axis, resulting in the production of glucocorticoids and catecholamines. A wide variety of immune cells express glucocorticoid receptors which bind cortisol and interfere with the function of NF-kB, regulating the production of cytokines (40).
Microglial cells have a long life span and can be activated for a long period of time which often links them to the underlying mechanism of the enduring effects of early-life stress exposure (41). Although activated microglia do not chronically produce cytokines or pro-inflammatory mediators, they overproduce cytokines once triggered by an immune insult leading to the development of neural disorders later in life (42,43). Compared with male rats, females show more activated microglia in the hippocampus, parietal cortex, and amygdala, which makes them more vulnerable to neuroinflammation during puberty (44) and perhaps more susceptible to certain brain disorders later in life.

Enduring Effect of Pubertal Neuroinflammation
Neuroinflammation is characterized by an amplified production of pro-inflammatory cytokines in the brain. When microglia and astrocytes are continuously activated, they can cause neural damage (45,46). Neuroinflammation underlies the development of various neurodegenerative diseases such as Alzheimer's and Parkinson's diseases (47). It also contributes to the development of mental disorders, such as anxiety, major depression, and post-traumatic stress disorder (48). Hence, adolescent exposure to environmental insults such as stress and inflammation can trigger the activation of microglia, leading to short-or long-term changes in immune function and increase in inflammatory cytokines and C-reactive proteins later in life (49,50). For example, in Wistar rats, repeated exposure to a viral mimetic immune challenge during puberty resulted in continued cognitive deficits at postnatal day 80 (51). In addition, long-term anxiety and depression-like behavior are observed in adult male and female mice respectively after pubertal/adolescent exposure to an immune challenge (52). However, the mechanism underlying these enduring effects remains unclear, one possibility could be due to lipopolysaccharide-induced injury to the hippocampus (53). These findings show that pubertal neuroinflammation whether it is acute (lasting for days), chronic (lasting for several weeks), subclinical (asymptomatic), or clinical (symptomatic) may cause permanent impairments in cognitive performance and behaviors. Thus, pubertal exposure to an immune challenge may lead to permanent injuries in specific areas of the brain and to long-term neurological disorders.

Microbiota Development With Emphasis on Puberty/Adolescence
Recent progress in next-generation microbial sequencing technology has allowed us to better understand the composition of the microbiota in the human gut and to investigate its structural changes across the life span (54).

Normal Gut Microbiota
The bacterial composition of infant microbiota is simple and unstable when compared with the adult microbiota which is highly diverse and fairly stable (55,56). Although the gut is not germfree while in utero, colonization of the infant gut occurs primarily during birth and the early postnatal period. Multiple factors contribute to gut colonization such as the mode of delivery, the place of birth, breastfeeding, and exposure to medication early in life (56). Recent findings show that adolescents have more diverse microbiota compared with adults (57). Adolescents have a higher abundance of Bifidobacterium and Clostridium compared with adults (57). Interestingly, gonadal steroid hormones cause sex differences in gut microbial composition. The gut microbiome is important for maintaining homeostasis; it is subject to significant shifts in composition and genetic content during critical periods of development and maturation. These changes occur during periods of dynamic brain development and are characterized by sex-specific shifts.
Males and females have distinct nutritional and energetic needs of growth, development, and reproduction which can explain the sex-specific shifts in the composition of the gut microbiome to meet these demands (58). For example, Actinobacteria and Tenericutes phyla and Allobaculum, Anaeroplasma, and Erwinia genera are more abundant in male than in female mice, whereas SMB 53 from the Clostridiaceae family and three members of family Lachnospiraceae are more abundant in female than in male mice (58,59). However, castration eliminates sex differences in intestinal microbiota of adult mice (60). It is unclear whether hormonal fluctuations during adolescence and their consequences on the microbiota of females and males contribute to the sex differences in the vulnerability to various disorders between males and females. It is known that autism and schizophrenia are more prevalent in males (61), whereas mood disorders and inflammatory bowel syndrome are more frequent in females (61,62). The immaturity of gut microbiota during adolescence could be vulnerable to environmental stressors. Hence, stress, infection, the use of antibiotics, poor diet, and inflammation can result in dysbiosis of the gut microbiota and may predispose the subject to brain disorders and psychiatric illnesses later in life (63,64). While the gut microbiome remains stable through adulthood, further changes in the microbiota occur in elderly individuals. Inadequate age-related changes in the microbiota may lead to a reduction in microbial diversity leading to inflammation ("inflammaging") and neurodegenerative diseases (65,66). Consequently, adolescence could be a time for crucial intervention to prevent microbial dysbiosis and optimize brain development and mental health later in life.

Microbiota and Neurodevelopment
The intestinal microbiota can influence the shaping of neural networks during neurodevelopment. For example, remodeling of synaptic connections and of neuronal brain circuits can be attributed to bacterial-derived molecules and impact neuronal function and behaviors (67). Work by Sudo and coworkers revealed an exaggerated stress response in germ-free mice or completely sterile mice when compared with specific pathogen-free counterparts (68) that have a normal gut flora. Remarkably, anxiety-like behavior in germ-free mice (sterile gut) can be reverted by conventionalization with specific pathogen-free microbiota. This effect is observed only in young mice but not at adult age (67,69) which highlights the key role of microbiota during neurodevelopment. Additionally, several proteins implicated in neuronal survival, differentiation, growth, synaptic plasticity, synaptic vesicles endocytosis, and synaptogenesis are expressed differently in germ-free mice. Although whole brain volume is not different in germ-free mice, they display larger amygdala and hippocampus volumes when compared with mice with conventional gut microbiota. These structural differences may be due to alterations in dendritic density and dendritic extensions and could be associated with neuropsychiatric disorders ranging from autism spectrum to anxiety disorders. Consequently, microbial alteration early in life can be associated with a number of neurodevelopmental disorders.

Microbiota and Stress
Stress can alter the gut microbial composition along the gastrointestinal tract. It favors the colonization by pathogenic bacterial species such as Citrobacter rodentium and increases cytokine concentration. Stress-induced gut dysbiosis may be due to alterations in the intestinal secretory IgA (70), leading to a dysfunctional gut-brain axis. Stress-induced activation of the HPA axis can affect thegut motility (71) and disrupt colonic homeostasis. The integrity of the intestinal barrier can also be compromised during stressful periods (72). However, exposure to probiotics can mitigate stress-induced activation of the HPA axis and intestinal permeability. For example, the probiotic Lactobacillus helveticus R0052 and Bifidobacterium longum R0175 can improve tight junction integrity in the colon of adult stressed mice and reduce bacterial translocation (73). These findings show that stress can cause a dysbiotic microbiota, which in turn can modulate brain biochemistry and mental health (74,75). Gut dysbiosis and age-related disorders with focus on neurodegeneration Because many neurodegenerative diseases start with enteric symptoms, gut dysbiosis is thought to be an important factor contributing to the initiation of these diseases with robust implication of the gutbrain axis (76).
Neurodegenerative diseases include disorders, such as Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), and amyloid lateral sclerosis (ALS). Older patients with neurodegenerative diseases show remarkable changes in their microbiota structure. For example, PD patients display less Prevotellaceae and anti-inflammatory butyrate-producing bacteria such as Roseburia and Faecalibacterium spp. and an increase in Enterobacteriaceae and pro-inflammatory Proteobacteria spp. In comparison to healthy aged subjects, Prevotellaceae are known to play a role in the protection of the intestinal barrier by producing mucin, whereas Enterobacteriaceae are associated with unstable posture (77,78). Moreover, PD patients display less short-chain fatty acids (SCFAs) as well as reduced concentration of butyrate involved in the protection of dopaminergic neurons (79). Interestingly, fecal microbiota transplant or transplantation of fecal bacteria from healthy donors into the intestine of a diseased recipient may help in the recovery and reduces PD symptoms in human patients as found per a single study case (80). However, transplantation of fecal samples from human PD patients into germ-free mice intensifies alpha-synuclein expression and leads to motor dysfunctions, while mice humanized with microbiota from matched healthy controls did not develop motor impairment (81). These findings suggest that the gut microbiota is directly related to the physiological symptoms of PD. Similarly, AD patients also display gut dysbiosis. AD patients have more gram-negative bacteria, which causes a disruption of the mucosal barrier. In addition, bacteria-producing amyloid β such as Bacillus subtilis, Klebsiella pneumonia, Mycobacterium spp., and Streptococcus spp. are further encountered in AD subjects (82). In AD patients, anti-inflammatory taxa (ie, Eubacterium rectale) are less abundant in the feces of participants, whereas proinflammatory taxa (ie, Escherichia and Shigella) that are linked to pro-inflammatory cytokines and amyloid deposition in the brain are more common (83). Together, these findings emphasize the important association between gut dysbiosis and neurodegenerative diseases and show the potential of targeting the gut microbiota as a therapeutic or preventative option.

Targeting Gut Microbiota in Neurodegenerative Disease and Potential Use of Probiotics
Modulating gut microbiome composition can improve the gastrointestinal barrier and reduce the development of inflammation and microglial activation, revealing a great therapeutic potential for the use of probiotics against neurodegeneration (84). In addition, targeting the microbiota composition can change the integrity of the blood-brain barrier (BBB) via producing different bacterial metabolites such as SCFAs. SCFAs are the main metabolites produced by bacterial fermentation of partially and nondigestible polysaccharides in the gastrointestinal tract; they are a major player in the maintenance of gut and immune homeostasis with anti-inflammatory, antitumorigenic, and antimicrobial effects properties (85). These molecules can be targeted by modifying the microbiome composition by using probiotics (86).
Another molecule that can be modified by probiotics is ferulic acid (FA). FA is a phenolic compound found in seeds of plants, vegetables, and fruits. It can be plentifully synthesized by some gut microbiota species such as L. fermentum NCIMB 5221 (87). It is a reactive oxygen species (ROS) scavenger and has anti-inflammatory properties (88). FA also has curative (able to cure) characteristics in neurodegeneration and cellular aging (89). FA stimulates neural stem cells proliferation by inducing neurogenesis and upregulating BDNF, a nerve growth factor (90). FA prevents Aβ aggregation and reduces Aβ fibril formation leading to a better AD behavioral phenotypes with improvement in memory formation (91). Histamine can as well be targeted by modulating gut microbiota. Different species such as Lactobacillus, Lactococcus, Streptococcus, Pediococcus, and Enterococcus (92) can secrete histamine. Certain probiotics, such as L. reuteri, exert their immunomodulatory activity and suppress TNFα via histamine production which decrease TLR signaling (93,94). Gut histamine is known to have a potential therapeutic effect in neurodegenerative diseases such as AD and multiple sclerosis (95). It acts as a neurotransmitter in the brain in addition to its role in immunomodulation, cell proliferation, and allergic reactions (96). It may induce either pro-inflammatory reactions by inducing allergy or anti-inflammatory responses by acting Figure 1. Microbiota diversity and neuronal complexity throughout life span with emphasis on the critical adolescence window. The core microbiota undergoes a dynamic shift across the life span. After initial colonization during infancy and birth, the gut microbiota continues to develop throughout childhood and adolescence. A less diverse microbiota is found in adolescent children in comparison with adults. As adulthood approaches, the gut microbiota stabilizes and becomes more diverse. With aging, microbiota diversity and stability decline. Shaping of the microbiota happens in parallel with neurodevelopment and they have similar critical developmental windows. Synaptogenesis begins at post-birth and synaptic density increases; the maximum density is attained at approximately 2 years of age. Then synaptic refinement and elimination occur in the human brain leading to reach the adult levels by mid-adolescence. During the critical developmental window (adolescence), there is an increased vulnerability to external stressors. Early adolescence is a key phase of neurodevelopment when major neuronal rewiring synaptic pruning or elimination of extra synapses occurs. A consequence of this important neuronal rewiring during adolescence is a high degree of vulnerability to different pathological stressors (inflammation, stress ….). The pubertal vulnerability of the brain to environmental stressors coincides with instability and immaturity of gut microbiota exacerbating the susceptibility of the brain to aberrant changes that may lead to the onset of brain disorders later in life (neurodegenerative or neurodevelopment disorders). on H4R receptors; distinct responses depend on the type of receptors involved (95,96).
Ghrelin is another biomolecule that impacts neurological function and can be modulated by targeting the microbiota. It acts as a satiety hormone and a neuropeptide in the CNS. It is produced when the stomach is empty to ease the hunger sensation. Ghrelin can regulate inflammation, neuromodulation, and energy homeostasis (97). Gut microbiota dynamics influence ghrelin production; its levels are decreased when the gut microbiota composition is altered (ie, alteration in populations such as Bifidobacterium spp.) (98). Ghrelin has some neuroprotective effects and plays a protective role in AD and PD, as it inhibits apoptosis, lessens ROS accumulation, improves synaptic plasticity, decreases the accumulation of Aβ, and reduces inflammation. Exposure to Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium bifidum, and Lactobacillus fermentum for 12 weeks improves the performance on the minimental state examination (MMSE), a cognitive test used to assess learning and memory (99).

Concluding Remarks and Future Directions
Puberty/adolescence is a fundamental and critical period of brain and microbiota development. During this period, the brain is extremely vulnerable to certain environmental factors such as stress and immune challenges and can result in enduring stress-induced mental illnesses and neurodegenerative disorders. Given the plasticity of the adolescent brain, the enduring effects of pubertal immune challenge on brain function, the role of the gut microbiota in neurodevelopment, and the important association between gut dysbiosis and neurodegenerative diseases, many preventive measures could be used to improve the resiliency of the teenage brain. For example, we could use a screening procedure to identify individuals at high risk for developing neurodegenerative diseases and expose them to prebiotics (health-promoting nondigestible food ingredients) and probiotics (health-promoting microbial food supplements) early in life to prevent the enduring effects of stress and inflammation. The gut-brain axis is an appealing target for the development of potential therapeutics for neurological diseases. However, further studies are needed to fully elucidate the impact of probiotics during adolescence in the prevention of debilitating diseases.

Funding
This publication was funded by a NSERC Collaborative Research and Development Grant (532223-18) to C.M. and by the Nutrition and Mental Health initiative at the Faculty of Health Sciences at the University of Ottawa.