Characterizing the human intestinal chondroitin sulfate glycosaminoglycan sulfation signature in inflammatory bowel disease.

The intestinal extracellular matrix (ECM) helps maintain appropriate tissue barrier function and regulate host-microbial interactions. Chondroitin sulfate- and dermatan sulfate-glycosaminoglycans (CS/DS-GAGs) are integral components of the intestinal ECM, and alterations in CS/DS-GAGs have been shown to significantly influence biological functions. Although pathologic ECM remodeling is implicated in inflammatory bowel disease (IBD), it is unknown whether changes in the intestinal CS/DS-GAG composition are also linked to IBD in humans. Our aim was to characterize changes in the intestinal ECM CS/DS-GAG composition in intestinal biopsy samples from patients with IBD using mass spectrometry. We characterized intestinal CS/DS-GAGs in 69 pediatric and young adult patients (n = 13 control, n = 32 active IBD, n = 24 IBD in remission) and 6 adult patients. Here, we report that patients with active IBD exhibit a significant decrease in the relative abundance of CS/DS isomers associated with matrix stability (CS-A and DS) compared to controls, while isomers implicated in matrix instability and inflammation (CS-C and CS-E) were significantly increased. This imbalance of intestinal CS/DS isomers was restored among patients in clinical remission. Moreover, the abundance of pro-stabilizing CS/DS isomers negatively correlated with clinical disease activity scores, whereas both pro-inflammatory CS-C and CS-E content positively correlated with disease activity scores. Thus, pediatric patients with active IBD exhibited increased pro-inflammatory and decreased pro-stabilizing CS/DS isomer composition, and future studies are needed to determine whether changes in the CS/DS-GAG composition play a pathogenic role in IBD.

Perineuronal net deglycosylation associates with tauopathy-induced gliosis and neurodegeneration.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by clinical symptoms of memory and cognitive deficiencies. Postmortem evaluation of AD brain tissue shows proteinopathy that closely associate with the progression of this dementing disorder, including the accumulation of extracellular beta amyloid (Aß) and intracellular hyperphosphorylated tau (pTau) with neurofibrillary tangles (NFTs). Current therapies targeting Aß have limited clinical efficacy and life-threatening side effects and highlight the need for alternative treatments targeting pTau and other pathophysiologic mechanisms driving AD pathogenesis. The brain's extracellular matrices (ECM), particularly perineuronal nets (PNNs), play a crucial role in brain functioning and neurocircuit stability, and reorganization of these unique PNN matrices has been associated with the progression of AD and accumulation of pTau in humans. We hypothesize that AD-associated changes in PNNs may in part be driven by the accumulation of pTau within the brain. In this work, we investigated whether the presence of pTau influenced PNN structural integrity and PNN chondroitin sulfate-glycosaminoglycan (CS-GAG) compositional changes in two transgenic mouse models expressing tauopathy-related AD pathology, PS19 (P301S) and Tau4RTg2652 mice. We show that PS19 mice exhibit an age-dependent loss of hippocampal PNN CS-GAGs, but not the underlying aggrecan core protein structures, in association with pTau accumulation, gliosis, and neurodegeneration. The loss of PNN CS-GAGs were linked to shifts in CS-GAG sulfation patterns to favor the neuroregenerative isomer, 2S6S-CS. Conversely, Tau4RTg2652 mice exhibit stable PNN structures and normal CS-GAG isomer composition despite robust pTau accumulation, suggesting a critical interaction between neuronal PNN glycan integrity and neighboring glial cell activation. Overall, our findings provide insights into the complex relationship between PNN CS-GAGs, pTau pathology, gliosis, and neurodegeneration in mouse models of tauopathy, and offer new therapeutic insights and targets for AD treatment.

High-fat diet feeding disrupts the coupling of thermoregulation to energy homeostasis.

OBJECTIVE: Preserving core body temperature across a wide range of ambient temperatures requires adaptive changes of thermogenesis that must be offset by corresponding changes of energy intake if body fat stores are also to be preserved. Among neurons implicated in the integration of thermoregulation with energy homeostasis are those that express both neuropeptide Y (NPY) and agouti-related protein (AgRP) (referred to herein as AgRP neurons). Specifically, cold-induced activation of AgRP neurons was recently shown to be required for cold exposure to increase food intake in mice. Here, we investigated how consuming a high-fat diet (HFD) impacts various adaptive responses to cold exposure as well as the responsiveness of AgRP neurons to cold. METHODS: To test this, we used immunohistochemistry, in vivo fiber photometry and indirect calorimetry for continuous measures of core temperature, energy expenditure, and energy intake in both chow- and HFD-fed mice housed at different ambient temperatures. RESULTS: We show that while both core temperature and the thermogenic response to cold are maintained normally in HFD-fed mice, the increase of energy intake needed to preserve body fat stores is blunted, resulting in weight loss. Using both immunohistochemistry and in vivo fiber photometry, we show that although cold-induced AgRP neuron activation is detected regardless of diet, the number of cold-responsive neurons appears to be blunted in HFD-fed mice. CONCLUSIONS: We conclude that HFD-feeding disrupts the integration of systems governing thermoregulation and energy homeostasis that protect body fat mass during cold exposure.

Warm Responsive Neurons in the Hypothalamic Preoptic Area are Potent Regulators of Glucose Homeostasis in Male Mice.

When mammals are exposed to a warm environment, overheating is prevented by activation of "warm-responsive" neurons (WRNs) in the hypothalamic preoptic area (POA) that reduce thermogenesis while promoting heat dissipation. Heat exposure also impairs glucose tolerance, but whether this also results from activation of POA WRNs is unknown. To address this question, we sought in the current work to determine if glucose intolerance induced by heat exposure can be attributed to activation of a specific subset of WRNs that express pituitary adenylate cyclase-activating peptide (ie, POAPacap neurons). We report that when mice are exposed to an ambient temperature sufficiently warm to activate POAPacap neurons, the expected reduction of energy expenditure is associated with glucose intolerance, and that these responses are recapitulated by chemogenetic POAPacap neuron activation. Because heat-induced glucose intolerance was not blocked by chemogenetic inhibition of POAPacap neurons, we conclude that POAPacap neuron activation is sufficient, but not required, to explain the impairment of glucose tolerance elicited by heat exposure.

Sustained inhibition of NPY/AgRP neuronal activity by FGF1.

In rodent models of type 2 diabetes (T2D), central administration of FGF1 normalizes elevated blood glucose levels in a manner that is sustained for weeks or months. Increased activity of NPY/AgRP neurons in the hypothalamic arcuate nucleus (ARC) is implicated in the pathogenesis of hyperglycemia in these animals, and the ARC is a key brain area for the antidiabetic action of FGF1. We therefore sought to determine whether FGF1 inhibits NPY/AgRP neurons and, if so, whether this inhibitory effect is sufficiently durable to offer a feasible explanation for sustained diabetes remission induced by central administration of FGF1. Here, we show that FGF1 inhibited ARC NPY/AgRP neuron activity, both after intracerebroventricular injection in vivo and when applied ex vivo in a slice preparation; we also showed that the underlying mechanism involved increased input from presynaptic GABAergic neurons. Following central administration, the inhibitory effect of FGF1 on NPY/AgRP neurons was also highly durable, lasting for at least 2 weeks. To our knowledge, no precedent for such a prolonged inhibitory effect exists. Future studies are warranted to determine whether NPY/AgRP neuron inhibition contributes to the sustained antidiabetic action elicited by intracerebroventricular FGF1 injection in rodent models of T2D.

Combined micro-osmotic pump infusion and intracerebroventricular injection to study FGF1 signaling pathways in the mouse brain.

Intracerebroventricular (icv) injection of fibroblast growth factor 1 (FGF1) elicits remission of diabetic hyperglycemia in rodent models of type 2 diabetes. Here, we present an optimized protocol to study the intracellular signaling pathways underlying the FGF1-induced sustained glucose lowering in the mouse brain. This protocol combines icv injection of FGF1 and osmotic mini-pump infusion of U0126, an inhibitor of MAPK/ERK signaling. We describe the surgical procedure and verification of U0126 inhibition of FGF1-stimulated hypothalamic MAPK/ERK signaling via western blot. For complete details on the use and execution of this protocol, please refer to Brown et al. (2021).

Decoding perineuronal net glycan sulfation patterns in the Alzheimer's disease brain.

The extracellular matrix (ECM) of the brain comprises unique glycan "sulfation codes" that influence neurological function. Perineuronal nets (PNNs) are chondroitin sulfate-glycosaminoglycan (CS-GAG) containing matrices that enmesh neural networks involved in memory and cognition, and loss of PNN matrices is reported in patients with neurocognitive and neuropsychiatric disorders including Alzheimer's disease (AD). Using liquid chromatography tandem mass spectrometry (LC-MS/MS), we show that patients with a clinical diagnosis of AD-related dementia undergo a re-coding of their PNN-associated CS-GAGs that correlates to Braak stage progression, hyperphosphorylated tau (p-tau) accumulation, and cognitive impairment. As these CS-GAG sulfation changes are detectable prior to the regional onset of classical AD pathology, they may contribute to the initiation and/or progression of the underlying degenerative processes and implicate the brain matrix sulfation code as a key player in the development of AD clinicopathology.

Role of hypothalamic MAPK/ERK signaling and central action of FGF1 in diabetes remission.

The capacity of the brain to elicit sustained remission of hyperglycemia in rodent models of type 2 diabetes following intracerebroventricular (icv) injection of fibroblast growth factor 1 (FGF1) is well established. Here, we show that following icv FGF1 injection, hypothalamic signaling by extracellular signal-regulated kinases 1 and 2 (ERK1/2), members of the mitogen-activated protein kinase (MAPK) family, is induced for at least 24 h. Further, we show that this prolonged response is required for the sustained antidiabetic action of FGF1 since it is abolished by sustained (but not acute) pharmacologic blockade of hypothalamic MAPK/ERK signaling. We also demonstrate that FGF1 R50E, a FGF1 mutant that activates FGF receptors but induces only transient hypothalamic MAPK/ERK signaling, fails to mimic the sustained glucose lowering induced by FGF1. These data identify sustained activation of hypothalamic MAPK/ERK signaling as playing an essential role in the mechanism underlying diabetes remission induced by icv FGF1 administration.

Leptin receptor neurons in the dorsomedial hypothalamus regulate diurnal patterns of feeding, locomotion, and metabolism.

The brain plays an essential role in driving daily rhythms of behavior and metabolism in harmony with environmental light-dark cycles. Within the brain, the dorsomedial hypothalamic nucleus (DMH) has been implicated in the integrative circadian control of feeding and energy homeostasis, but the underlying cell types are unknown. Here, we identify a role for DMH leptin receptor-expressing (DMHLepR) neurons in this integrative control. Using a viral approach, we show that silencing neurotransmission in DMHLepR neurons in adult mice not only increases body weight and adiposity but also phase-advances diurnal rhythms of feeding and metabolism into the light cycle and abolishes the normal increase in dark-cycle locomotor activity characteristic of nocturnal rodents. Finally, DMHLepR-silenced mice fail to entrain to a restrictive change in food availability. Together, these findings identify DMHLepR neurons as critical determinants of the daily time of feeding and associated metabolic rhythms.

Cold-induced hyperphagia requires AgRP neuron activation in mice.

To maintain energy homeostasis during cold exposure, the increased energy demands of thermogenesis must be counterbalanced by increased energy intake. To investigate the neurobiological mechanisms underlying this cold-induced hyperphagia, we asked whether agouti-related peptide (AgRP) neurons are activated when animals are placed in a cold environment and, if so, whether this response is required for the associated hyperphagia. We report that AgRP neuron activation occurs rapidly upon acute cold exposure, as do increases of both energy expenditure and energy intake, suggesting the mere perception of cold is sufficient to engage each of these responses. We further report that silencing of AgRP neurons selectively blocks the effect of cold exposure to increase food intake but has no effect on energy expenditure. Together, these findings establish a physiologically important role for AgRP neurons in the hyperphagic response to cold exposure.

Distinct Neuronal Projections From the Hypothalamic Ventromedial Nucleus Mediate Glycemic and Behavioral Effects.

The hypothalamic ventromedial nucleus (VMN) is implicated both in autonomic control of blood glucose and in behaviors including fear and aggression, but whether these divergent effects involve the same or distinct neuronal subsets and their projections is unknown. To address this question, we used an optogenetic approach to selectively activate the subset of VMN neurons that express neuronal nitric oxide synthase 1 (VMNNOS1 neurons) implicated in glucose counterregulation. We found that photoactivation of these neurons elicits 1) robust hyperglycemia achieved by activation of counterregulatory responses usually reserved for the physiological response to hypoglycemia and 2) defensive immobility behavior. Moreover, we show that the glucagon, but not corticosterone, response to insulin-induced hypoglycemia is blunted by photoinhibition of the same neurons. To investigate the neurocircuitry by which VMNNOS1 neurons mediate these effects, and to determine whether these diverse effects are dissociable from one another, we activated downstream VMNNOS1 projections in either the anterior bed nucleus of the stria terminalis (aBNST) or the periaqueductal gray (PAG). Whereas glycemic responses are fully recapitulated by activation of VMNNOS1 projections to the aBNST, freezing immobility occurred only upon activation of VMNNOS1 terminals in the PAG. These findings support previous evidence of a VMN→aBNST neurocircuit involved in glucose counterregulation and demonstrate that activation of VMNNOS1 neuronal projections supplying the PAG robustly elicits defensive behaviors.