Assessing translational applicability of perineuronal net dysfunction in Alzheimer's disease across species.

In the context of aging and age-associated neurodegenerative disorders, the brain's extracellular matrix (ECM) serves as a critical regulator for neuronal health and cognitive function. Within the extracellular space, proteoglycans and their glycosaminoglycan attachments play essential roles in forming, stabilizing, and protecting neural circuits throughout neurodevelopment and adulthood. Recent studies in rodents reveal that chondroitin sulfate-glycosaminoglycan (CS-GAG) containing perineuronal nets (PNNs) exhibit both structural and compositional differences throughout the brain. While animal studies are illuminating, additional research is required to translate these interregional PNN/CS-GAG variations to human brain tissue. In this perspective article, we first investigate the translational potential for interregional CS-GAG variances across species as novel targets for region-specific therapeutic development. We specifically focus on the observation that alterations in brain PNN-associated CS-GAGs have been linked with the progression of Alzheimer's disease (AD) neuropathology in humans, but these changes have not been fully recapitulated in rodent models of this disease. A second highlight of this perspective article investigates whether AD-associated shifts in CS-GAGs in humans may be dependent on region-specific baseline differences in CS-GAG sulfation patterning. The current findings begin to disentangle the intricate relationships between the interregional differences in brain PNN/CS-GAG matrices across species, while emphasizing the need to better understand the close relationship between dementia and changes in brain CS-GAG sulfation patterns in patients with AD and related dementias.

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.

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.

A versatile pumpless multi-channel fluidics system for maintenance and real-time functional assessment of tissue and cells.

To address the needs of the life sciences community and the pharmaceutical industry in pre-clinical drug development to both maintain and continuously assess tissue metabolism and function with simple and rapid systems, we improved on the initial BaroFuse to develop it into a fully functional, pumpless, scalable multi-channel fluidics instrument that continuously measures changes in oxygen consumption and other endpoints in response to test compounds. We and several other laboratories assessed it with a wide range of tissue types including retina, pancreatic islets, liver, and hypothalamus with both aqueous and gaseous test compounds. The setup time was less than an hour for all collaborating groups, and there was close agreement between data obtained from the different laboratories. This easy-to-use system reliably generates real-time metabolic and functional data from tissue and cells in response to test compounds that will address a critical need in basic and applied research.

Diabetes exacerbates inflammatory bowel disease in mice with diet-induced obesity.

BACKGROUND: The increased prevalence of inflammatory bowel disease (IBD) among patients with obesity and type 2 diabetes suggests a causal link between these diseases, potentially involving the effect of hyperglycemia to disrupt intestinal barrier integrity. AIM: To investigate whether the deleterious impact of diabetes on the intestinal barrier is associated with increased IBD severity in a murine model of colitis in mice with and without diet-induced obesity. METHODS: Mice were fed chow or a high-fat diet and subsequently received streptozotocin to induce diabetic-range hyperglycemia. Six weeks later, dextran sodium sulfate was given to induce colitis. In select experiments, a subset of diabetic mice was treated with the antidiabetic drug dapagliflozin prior to colitis onset. Endpoints included both clinical and histological measures of colitis activity as well as histochemical markers of colonic epithelial barrier integrity. RESULTS: In mice given a high-fat diet, but not chow-fed animals, diabetes was associated with significantly increased clinical colitis activity and histopathologic markers of disease severity. Diabetes was also associated with a decrease in key components that regulate colonic epithelial barrier integrity (colonic mucin layer content and epithelial tight junction proteins) in diet-induced obese mice. Each of these effects of diabetes in diet-induced obese mice was ameliorated by restoring normoglycemia. CONCLUSION: In obese mice, diabetes worsened clinical and pathologic outcomes of colitis via mechanisms that are reversible with treatment of hyperglycemia. Hyperglycemia-induced intestinal barrier dysfunction offers a plausible mechanism linking diabetes to increased colitis severity. These findings suggest that effective diabetes management may decrease the clinical severity of IBD.

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.

Identification of Hypothalamic Glucoregulatory Neurons That Sense and Respond to Changes in Glycemia.

To investigate whether glucoregulatory neurons in the hypothalamus can sense and respond to physiological variation in the blood glucose (BG) level, we combined continuous arterial glucose monitoring with continuous measures of the activity of a specific subset of neurons located in the hypothalamic ventromedial nucleus that express pituitary adenylate cyclase activating peptide (VMNPACAP neurons) obtained using fiber photometry. Data were collected in conscious, free-living mice during a 1-h baseline monitoring period and a subsequent 2-h intervention period during which the BG level was raised either by consuming a chow or a high-sucrose meal or by intraperitoneal glucose injection. Cross-correlation analysis revealed that, following a 60- to 90-s delay, interventions that raise the BG level reliably associate with reduced VMNPACAP neuron activity (P < 0.01). In addition, a strong positive correlation between BG and spontaneous VMNPACAP neuron activity was observed under basal conditions but with a much longer (∼25 min) temporal offset, consistent with published evidence that VMNPACAP neuron activation raises the BG level. Together, these findings are suggestive of a closed-loop system whereby VMNPACAP neuron activation increases the BG level; detection of a rising BG level, in turn, feeds back to inhibit these neurons. To our knowledge, these findings constitute the first evidence of a role in glucose homeostasis for glucoregulatory neurocircuits that, like pancreatic ß-cells, sense and respond to physiological variation in glycemia. ARTICLE HIGHLIGHTS: By combining continuous arterial glucose monitoring with fiber photometry, studies investigated whether neurons in the murine ventromedial nucleus that express pituitary adenylate cyclase activating peptide (VMNPACAP neurons) detect and respond to changes in glycemia in vivo. VMNPACAP neuron activity rapidly decreases (within <2 min) when the blood glucose level is raised by either food consumption or glucose administration. Spontaneous VMNPACAP neuron activity also correlates positively with glycemia, but with a longer temporal offset, consistent with reports that hyperglycemia is induced by experimental activation of these neurons. Like pancreatic ß-cells, neurons in the hypothalamic ventromedial nucleus appear to sense and respond to physiological variation in glycemia.

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.

The "Loss" of Perineuronal Nets in Alzheimer's Disease: Missing or Hiding in Plain Sight?

Perineuronal nets (PNNs) are chondroitin-sulfate glycosaminoglycan (CS-GAG) containing extracellular matrix structures that assemble around neurons involved in learning, memory, and cognition. Owing to the unique patterning of negative charges stemming from sulfate modifications to the attached CS-GAGs, these matrices play key roles in mediating glycan-protein binding, signaling interactions, and charged ion buffering of the underlying circuitry. Histochemical loss of PNN matrices has been reported for a range of neurocognitive and neurodegenerative diseases, implying that PNNs might be a key player in the pathogenesis of neurological disorders. In this hypothesis and theory article, we begin by highlighting PNN changes observed in human postmortem brain tissue associated with Alzheimer's disease (AD) and corresponding changes reported in rodent models of AD neuropathology. We then discuss the technical limitations surrounding traditional methods for PNN analyses and propose alternative explanations to these historical findings. Lastly, we embark on a global re-evaluation of the interpretations for PNN changes across brain regions, across species, and in relation to other neurocognitive disorders.

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).

Neurogenin-3 Enteric Endocrinopathy: A Rare Case of Pediatric Congenital Diarrhea and Diabetes Mellitus.

Disorders of intestinal enteroendocrine cells (EEC) are a rare cause of congenital diarrhea and diabetes. The gene NEUROG3 is essential in EEC differentiation, and mutations in this gene lead to a paucity of EEC in the intestine and pancreas, often presenting clinically as congenital diarrhea and diabetes mellitus. We present the earliest known diagnosis of NEUROG3-associated enteric endocrinopathy, which was identified on a neonatal diabetes genetic panel sent at 4 weeks of age. Our patient presented with severe diarrhea, malnutrition, electrolyte derangements, and neonatal diabetes. He was started on parenteral nutrition at 3 months of age for nutritional and hydration support and required long-acting insulin for his diabetes. We demonstrate significant reduction in EEC, including cells expressing glucagon-like peptide-1, in intestinal biopsies from our patient, raising the possibility that loss of glucagon-like peptide-1 contributes to NEUROG3-associated diarrhea and diabetes mellitus. This case advances our understanding of the presentation, diagnosis, and management of this rare disease.

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.

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.

Transcriptomic analysis links diverse hypothalamic cell types to fibroblast growth factor 1-induced sustained diabetes remission.

In rodent models of type 2 diabetes (T2D), sustained remission of hyperglycemia can be induced by a single intracerebroventricular (icv) injection of fibroblast growth factor 1 (FGF1), and the mediobasal hypothalamus (MBH) was recently implicated as the brain area responsible for this effect. To better understand the cellular response to FGF1 in the MBH, we sequenced >79,000 single-cell transcriptomes from the hypothalamus of diabetic Lepob/ob mice obtained on Days 1 and 5 after icv injection of either FGF1 or vehicle. A wide range of transcriptional responses to FGF1 was observed across diverse hypothalamic cell types, with glial cell types responding much more robustly than neurons at both time points. Tanycytes and ependymal cells were the most FGF1-responsive cell type at Day 1, but astrocytes and oligodendrocyte lineage cells subsequently became more responsive. Based on histochemical and ultrastructural evidence of enhanced cell-cell interactions between astrocytes and Agrp neurons (key components of the melanocortin system), we performed a series of studies showing that intact melanocortin signaling is required for the sustained antidiabetic action of FGF1. These data collectively suggest that hypothalamic glial cells are leading targets for the effects of FGF1 and that sustained diabetes remission is dependent on intact melanocortin signaling.

Hypothalamic perineuronal net assembly is required for sustained diabetes remission induced by fibroblast growth factor 1 in rats.

We recently showed that perineuronal nets (PNNs) enmesh glucoregulatory neurons in the arcuate nucleus (Arc) of the mediobasal hypothalamus (MBH)1, but whether these PNNs play a role in either the pathogenesis of type 2 diabetes (T2D) or its treatment remains unclear. Here we show that PNN abundance within the Arc is markedly reduced in the Zucker diabetic fatty (ZDF) rat model of T2D, compared with normoglycaemic rats, correlating with altered PNN-associated sulfation patterns of chondroitin sulfate glycosaminoglycans in the MBH. Each of these PNN-associated changes is reversed following a single intracerebroventricular (icv) injection of fibroblast growth factor 1 (FGF1) at a dose that induces sustained diabetes remission in male ZDF rats. Combined with previous work localizing this FGF1 effect to the Arc area2-4, our finding that enzymatic digestion of Arc PNNs markedly shortens the duration of diabetes remission following icv FGF1 injection in these animals identifies these extracellular matrix structures as previously unrecognized participants in the mechanism underlying diabetes remission induced by the central action of FGF1.

Perineuronal Net Formation during the Critical Period for Neuronal Maturation in the Hypothalamic Arcuate Nucleus.

In leptin-deficient ob/ob mice, obesity and diabetes are associated with abnormal development of neurocircuits in the hypothalamic arcuate nucleus (ARC)1, a critical brain area for energy and glucose homeostasis2,3. As this developmental defect can be remedied by systemic leptin administration, but only if given before postnatal day 28, a critical period (CP) for leptin-dependent development of ARC neurocircuits has been proposed4. In other brain areas, CP closure coincides with the appearance of perineuronal nets (PNNs), extracellular matrix specializations that restrict the plasticity of neurons that they enmesh5. Here we report that in humans as well as rodents, subsets of neurons in the mediobasal aspect of the ARC are enmeshed by PNN-like structures. In mice, these neurons are densely-packed into a continuous ring that encircles the junction of the ARC and median eminence, which facilitates exposure of ARC neurons to the circulation. Most of the enmeshed neurons are both GABAergic and leptin receptor-positive, including a majority of Agrp neurons. Postnatal formation of the PNN-like structures coincides precisely with closure of the CP for Agrp neuron maturation and is dependent on input from circulating leptin, as postnatal ob/ob mice have reduced ARC PNN-like material that is restored by leptin administration during the CP. We conclude that neurons crucial to metabolic homeostasis are enmeshed by PNN-like structures and organized into a densely packed cluster situated circumferentially at the ARC-ME junction, where metabolically-relevant humoral signals are sensed.

Plasma Fibroblast Growth Factor 21 Is Associated with Subsequent Growth in a Cohort of Underweight Children in Bangladesh.

BACKGROUND: Current nutritional intervention strategies have not proven effective in improving childhood ponderal and linear growth in underweight and stunted children. Novel markers are needed to classify children who are likely to respond to available interventions and to identify those requiring additional interventions. Fibroblast Growth Factor 21 (FGF21), an endocrine hormone that regulates metabolism and growth during periods of reduced protein intake, may be useful in this context. OBJECTIVES: We aimed to determine the associations between plasma FGF21 concentrations and subsequent growth, and the association between change in FGF21 concentrations and concurrent growth, in children receiving nutritional supplementation. METHODS: A total of 120 children between ages 6 and 13 mo with weight-for-age z score (WAZ) between -3 and -2 were enrolled from an urban slum in Dhaka, Bangladesh. Children received 376-kcal feeding supplements daily for 5 mo and were followed for 5 additional mo. FGF21 was measured in plasma collected at enrollment and month 5. FGF21 values that fell above the 90th percentile of baseline concentrations (1056.5 pg/mL) were considered high. Linear regression was used to examine the association between baseline FGF21 status and 5-mo change in WAZ and length-for-age z score (LAZ), and the association between 5-mo change in FGF21 and concurrent WAZ and LAZ change. RESULTS: The median baseline FGF21 concentration was 241.4 pg/mL (IQR: 111.7, 451.3 pg/mL). On average, children with high baseline FGF21 gained 0.58 WAZ (95% CI: 0.28, 0.88) and 0.54 LAZ (95% CI: 0.23, 0.84) more during supplementation than those with low values. Change in FGF21 concentration during supplementation was negatively associated with change in WAZ (-0.48; 95% CI: -0.67, -0.29) and LAZ (-0.31; 95% CI: -0.52, -0.11). CONCLUSIONS: FGF21 may be a useful marker of growth faltering and may allow identification of children who are more or less likely to respond to nutritional supplementation. This trial was registered at clinicaltrials.gov as NCT02441426.