The 5:2 diet does not increase adult hippocampal neurogenesis or enhance spatial memory in mice.

New neurones are generated throughout life in the mammalian brain in a process known as adult hippocampal neurogenesis (AHN). Since this phenomenon grants a high degree of neuroplasticity influencing learning and memory, identifying factors that regulate AHN may be important for ameliorating age-related cognitive decline. Calorie restriction (CR) has been shown to enhance AHN and improve memory, mediated by the stomach hormone, ghrelin. Intermittent fasting (IF), a dietary strategy offering more flexibility than conventional CR, has also been shown to promote aspects of AHN. The 5:2 diet is a popular form of IF; however, its effects on AHN are not well characterised. To address this, we quantified AHN in adolescent and adult wild-type and ghrelin-receptor-deficient mice following 6 weeks on a 5:2 diet. We report an age-related decline in neurogenic processes. However, the 5:2 diet does not increase AHN nor enhance memory performance, suggesting that this specific form of IF is ineffective in promoting brain plasticity to support learning.

Acyl-Ghrelin Attenuates Neurochemical and Motor Deficits in the 6-OHDA Model of Parkinson's Disease.

The feeding-related hormone, acyl-ghrelin, protects dopamine neurones in murine 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-based models of experimental Parkinson's disease (PD). However, the potential protective effect of acyl-ghrelin on substantia nigra pars compacta (SNpc) dopaminergic neurones and consequent behavioural correlates in the more widely used 6-hydroxydopamine (6-OHDA) rat medial forebrain bundle (MFB) lesion model of PD are unknown. To address this question, acyl-ghrelin levels were raised directly by mini-pump infusion for 7 days prior to unilateral injection of 6-OHDA into the MFB with assessment of amphetamine-induced rotations on days 27 and 35, and immunohistochemical analysis of dopaminergic neurone survival. Whilst acyl-ghrelin treatment was insufficient to elevate food intake or body weight, it attenuated amphetamine-induced circling behaviour and SNpc dopamine neurone loss induced by 6-OHDA. These data support the notion that elevating circulating acyl-ghrelin may be a valuable approach to slow or impair progression of neurone loss in PD.

Acylation, a Conductor of Ghrelin Function in Brain Health and Disease.

Acyl-ghrelin (AG) is an orexigenic hormone that has a unique octanoyl modification on its third serine residue. It is often referred to as the "hunger hormone" due to its involvement in stimulating food intake and regulating energy homeostasis. The discovery of the enzyme ghrelin-O-acyltransferase (GOAT), which catalyses ghrelin acylation, provided further insights into the relevance of this lipidation process for the activation of the growth hormone secretagogue receptor (GHS-R) by acyl-ghrelin. Although acyl-ghrelin is predominantly linked with octanoic acid, a range of saturated fatty acids can also bind to ghrelin possibly leading to specific functions. Sources of ghrelin acylation include beta-oxidation of longer chain fatty acids, with contributions from fatty acid synthesis, the diet, and the microbiome. In addition, both acyl-ghrelin and unacyl-ghrelin (UAG) have feedback effects on lipid metabolism which in turn modulate their levels. Recently we showed that whilst acyl-ghrelin promotes adult hippocampal neurogenesis and enhances memory function, UAG inhibits these processes. As a result, we postulated that the circulating acyl-ghrelin:unacyl-ghrelin (AG:UAG) ratio might be an important regulator of neurogenesis and cognition. In this review, we discuss emerging evidence behind the relevance of ghrelin acylation in the context of brain physiology and pathology, as well as the current challenges of identifying the provenance of the acyl moiety.

Metabolic hormones mediate cognition.

Recent biochemical and behavioural evidence indicates that metabolic hormones not only regulate energy intake and nutrient content, but also modulate plasticity and cognition in the central nervous system. Disruptions in metabolic hormone signalling may provide a link between metabolic syndromes like obesity and diabetes, and cognitive impairment. For example, altered metabolic homeostasis in obesity is a strong determinant of the severity of age-related cognitive decline and neurodegenerative disease. Here we review the evidence that eating behaviours and metabolic hormones-particularly ghrelin, leptin, and insulin-are key players in the delicate regulation of neural plasticity and cognition. Caloric restriction and antidiabetic therapies, both of which affect metabolic hormone levels can restore metabolic homeostasis and enhance cognitive function. Thus, metabolic hormone pathways provide a promising target for the treatment of cognitive decline.

Ghrelin Acylation-A Post-Translational Tuning Mechanism Regulating Adult Hippocampal Neurogenesis.

Adult hippocampal neurogenesis-the generation of new functional neurones in the adult brain-is impaired in aging and many neurodegenerative disorders. We recently showed that the acylated version of the gut hormone ghrelin (acyl-ghrelin) stimulates adult hippocampal neurogenesis while the unacylated form of ghrelin inhibits it, thus demonstrating a previously unknown function of unacyl-ghrelin in modulating hippocampal plasticity. Analysis of plasma samples from Parkinson's disease patients with dementia demonstrated a reduced acyl-ghrelin:unacyl-ghrelin ratio compared to both healthy controls and cognitively intact Parkinson's disease patients. These data, from mouse and human studies, suggest that restoring acyl-ghrelin signalling may promote the activation of pathways to support memory function. In this short review, we discuss the evidence for ghrelin's role in regulating adult hippocampal neurogenesis and the enzymes involved in ghrelin acylation and de-acylation as targets to treat mood-related disorders and dementia.

Ghrelin mediated hippocampal neurogenesis.

The stomach hormone, ghrelin, which is released during food restriction, provides a link between circulating energy state and adaptive brain function. The maintenance of such homeostatic systems is essential for an organism to survive and thrive, and accumulating evidence points to ghrelin being a key regulator of adult hippocampal neurogenesis and memory function. Aberrant neurogenesis is linked to cognitive decline in aging and neurodegeneration. Therefore, identifying endogenous metabolic factors that regulate new adult-born neuron formation is an important objective in understanding the link between nutritional status and CNS function. Here, we review current developments in our understanding of ghrelin's role in regulating neurogenesis and memory function.

Analysis of target mRNAs in the fixed-frozen human brain using a modified BaseScope-ISH Assay protocol.

We describe a modified BaseScope™ Assay protocol (ACDBio) for RNA in situ hybridization on fixed-frozen human brain tissue. The original protocol caused tissue detachment due to harsh tissue pre-treatment. We therefore optimized it to improve tissue stability while providing high stain quality in fragile post-mortem tissue from aged donors with advanced neurodegeneration. The main changes include two additional fixation steps and modifications to the pre-treatment protocol. We also describe tissue imaging and stain quantification using the open-source QuPath software. For complete details on the use and execution of this protocol, please refer to Hornsby et al. (2020).

L-dopa-Dependent Effects of GLP-1R Agonists on the Survival of Dopaminergic Cells Transplanted into a Rat Model of Parkinson Disease.

Cell therapy is a promising treatment for Parkinson's disease (PD), however clinical trials to date have shown relatively low survival and significant patient-to-patient variability. Glucagon Like Peptide-1 receptor (GLP-1R) agonists have potential neuroprotective effects on endogenous dopaminergic neurons. This study explores whether these agents could similarly support the growth and survival of newly transplanted neurons. 6-OHDA lesioned Sprague Dawley rats received intra-striatal grafts of dopaminergic ventral mesencephalic cells from embryonic day 14 Wistar rat embryos. Transplanted rats then received either saline or L-dopa (12 mg/kg) administered every 48 h prior to, and following cell transplantation. Peripheral GLP-1R agonist administration (exendin-4, 0.5 µg/kg twice daily or liraglutide, 100 µg/kg once daily) commenced immediately after cell transplantation and was maintained throughout the study. Graft survival increased under administration of exendin-4, with motor function improving significantly following treatment with both exendin-4 and liraglutide. However, this effect was not observed in rats administered with L-dopa. In contrast, L-dopa treatment with liraglutide increased graft volume, with parallel increases in motor function. However, this improvement was accompanied by an increase in leukocyte infiltration around the graft. The co-administration of L-dopa and exendin-4 also led to indicators of insulin resistance not seen with liraglutide, which may underpin the differential effects observed between the two GLP1-R agonists. Overall, there may be some benefit to the supplementation of grafted patients with GLP-1R agonists but the potential interaction with other pharmacological treatments needs to be considered in more depth.

MRI-guided histology of TDP-43 knock-in mice implicates parvalbumin interneuron loss, impaired neurogenesis and aberrant neurodevelopment in amyotrophic lateral sclerosis-frontotemporal dementia.

Amyotrophic lateral sclerosis and frontotemporal dementia are overlapping diseases in which MRI reveals brain structural changes in advance of symptom onset. Recapitulating these changes in preclinical models would help to improve our understanding of the molecular causes underlying regionally selective brain atrophy in early disease. We therefore investigated the translational potential of the TDP-43Q331K knock-in mouse model of amyotrophic lateral sclerosis-frontotemporal dementia using MRI. We performed in vivo MRI of TDP-43Q331K knock-in mice. Regions of significant volume change were chosen for post-mortem brain tissue analyses. Ex vivo computed tomography was performed to investigate skull shape. Parvalbumin neuron density was quantified in post-mortem amyotrophic lateral sclerosis frontal cortex. Adult mutants demonstrated parenchymal volume reductions affecting the frontal lobe and entorhinal cortex in a manner reminiscent of amyotrophic lateral sclerosis-frontotemporal dementia. Subcortical, cerebellar and brain stem regions were also affected in line with observations in pre-symptomatic carriers of mutations in C9orf72, the commonest genetic cause of both amyotrophic lateral sclerosis and frontotemporal dementia. Volume loss was also observed in the dentate gyrus of the hippocampus, along with ventricular enlargement. Immunohistochemistry revealed reduced parvalbumin interneurons as a potential cellular correlate of MRI changes in mutant mice. By contrast, microglia was in a disease activated state even in the absence of brain volume loss. A reduction in immature neurons was found in the dentate gyrus, indicative of impaired adult neurogenesis, while a paucity of parvalbumin interneurons in P14 mutant mice suggests that TDP-43Q331K disrupts neurodevelopment. Computerized tomography imaging showed altered skull morphology in mutants, further suggesting a role for TDP-43Q331K in development. Finally, analysis of human post-mortem brains confirmed a paucity of parvalbumin interneurons in the prefrontal cortex in sporadic amyotrophic lateral sclerosis and amyotrophic lateral sclerosis linked to C9orf72 mutations. Regional brain MRI changes seen in human amyotrophic lateral sclerosis-frontotemporal dementia are recapitulated in TDP-43Q331K knock-in mice. By marrying in vivo imaging with targeted histology, we can unravel cellular and molecular processes underlying selective brain vulnerability in human disease. As well as helping to understand the earliest causes of disease, our MRI and histological markers will be valuable in assessing the efficacy of putative therapeutics in TDP-43Q331K knock-in mice.

Automated Quantification of Mitochondrial Fragmentation in an In Vitro Parkinson's Disease Model.

Neuronal mitochondrial fragmentation is a phenotype exhibited in models of neurodegeneration such as Parkinson's disease. Delineating the dysfunction in mitochondrial dynamics found in diseased states can aid our understanding of underlying mechanisms of disease progression and possibly identify novel therapeutic approaches. Advances in microscopy and the availability of intuitive open-access software have accelerated the rate of image acquisition and analysis, respectively. These developments allow routine biology researchers to rapidly turn hypotheses into results. In this protocol, we describe the utilization of cell culture techniques, high-content imaging (HCI), and the subsequent open-source image analysis pipeline for the quantification of mitochondrial fragmentation in the context of a rotenone-based in vitro Parkinson's disease model. © 2020 The Authors. Basic Protocol 1: SN4741 neuron culture and treatment in a rotenone-based model of Parkinson's disease Basic Protocol 2: Identification of cell nuclei, measurement of mitochondrial membrane potential, and measurement of mitochondrial fragmentation in mouse-derived midbrain dopaminergic neurons.

Unacylated-Ghrelin Impairs Hippocampal Neurogenesis and Memory in Mice and Is Altered in Parkinson's Dementia in Humans.

Blood-borne factors regulate adult hippocampal neurogenesis and cognition in mammals. We report that elevating circulating unacylated-ghrelin (UAG), using both pharmacological and genetic methods, reduced hippocampal neurogenesis and plasticity in mice. Spatial memory impairments observed in ghrelin-O-acyl transferase-null (GOAT-/-) mice that lack acyl-ghrelin (AG) but have high levels of UAG were rescued by acyl-ghrelin. Acyl-ghrelin-mediated neurogenesis in vitro was dependent on non-cell-autonomous BDNF signaling that was inhibited by UAG. These findings suggest that post-translational acylation of ghrelin is important to neurogenesis and memory in mice. To determine relevance in humans, we analyzed circulating AG:UAG in Parkinson disease (PD) patients diagnosed with dementia (PDD), cognitively intact PD patients, and controls. Notably, plasma AG:UAG was only reduced in PDD. Hippocampal ghrelin-receptor expression remained unchanged; however, GOAT+ cell number was reduced in PDD. We identify UAG as a regulator of hippocampal-dependent plasticity and spatial memory and AG:UAG as a putative circulating diagnostic biomarker of dementia.

Ghrelin-Mediated Hippocampal Neurogenesis: Implications for Health and Disease.

There is a close relationship between cognition and nutritional status, however, the mechanisms underlying this relationship require elucidation. The stomach hormone, ghrelin, which is released during food restriction, provides a link between circulating energy state and adaptive brain function. The maintenance of such homeostatic systems is essential for an organism to thrive and survive, and accumulating evidence points to ghrelin being key in promoting adult hippocampal neurogenesis and memory. Aberrant neurogenesis is linked to cognitive decline in ageing and neurodegeneration. Therefore, identifying endogenous metabolic factors that regulate new adult-born neurone formation is an important objective in understanding the link between nutritional status and central nervous system (CNS) function. Here, we review current developments in our understanding of ghrelin's role in regulating neurogenesis and memory function.

Calorie restriction activates new adult born olfactory-bulb neurones in a ghrelin-dependent manner but acyl-ghrelin does not enhance subventricular zone neurogenesis.

The ageing and degenerating brain show deficits in neural stem/progenitor cell (NSPC) plasticity that are accompanied by impairments in olfactory discrimination. Emerging evidence suggests that the gut hormone ghrelin plays an important role in protecting neurones, promoting synaptic plasticity and increasing hippocampal neurogenesis in the adult brain. In the present study, we investigated the role of ghrelin with respect to modulating adult subventricular zone (SVZ) NSPCs that give rise to new olfactory bulb (OB) neurones. We characterised the expression of the ghrelin receptor, growth hormone secretagogue receptor (GHSR), using an immunohistochemical approach in GHSR-eGFP reporter mice to show that GHSR is expressed in several regions, including the OB but not in the SVZ of the lateral ventricle. These data suggest that acyl-ghrelin does not mediate a direct effect on NSPC in the SVZ. Consistent with these findings, treatment with acyl-ghrelin or genetic silencing of GHSR did not alter NSPC proliferation within the SVZ. Similarly, using a bromodeoxyuridine pulse-chase approach, we show that peripheral treatment of adult rats with acyl-ghrelin did not increase the number of new adult-born neurones in the granule cell layer of the OB. These data demonstrate that acyl-ghrelin does not increase adult OB neurogenesis. Finally, we investigated whether elevating ghrelin indirectly, via calorie restriction (CR), regulated the activity of new adult-born cells in the OB. Overnight CR induced c-Fos expression in new adult-born OB cells but not in developmentally born cells, whereas neuronal activity was absent following re-feeding. These effects were not present in ghrelin-/- mice, suggesting that adult-born cells are uniquely sensitive to changes in ghrelin mediated by fasting and re-feeding. In summary, ghrelin does not promote neurogenesis in the SVZ and OB; however, new adult-born OB cells are activated by CR in a ghrelin-dependent manner.

Ghrelin mediated neuroprotection - A possible therapy for Parkinson's disease?

Parkinson's disease is a common age-related neurodegenerative disorder affecting 10 million people worldwide, but the mechanisms underlying its pathogenesis are still unclear. The disease is characterised by dopamine nerve cell loss in the mid-brain and intra-cellular accumulation of α-synuclein that results in motor and non-motor dysfunction. In this review, we discuss the neuroprotective effects of the stomach hormone, ghrelin, in models of Parkinson's disease. Recent findings suggest that it may modulate mitochondrial function and autophagic clearance of impaired organelle in response to changes in cellular energy balance. We consider the putative cellular mechanisms underlying ghrelin-action and the possible role of ghrelin mimetics in slowing or preventing Parkinson's disease progression. This article is part of the Special Issue entitled 'Metabolic Impairment as Risk Factors for Neurodegenerative Disorders.'

Unacylated ghrelin promotes adipogenesis in rodent bone marrow via ghrelin O-acyl transferase and GHS-R1a activity: evidence for target cell-induced acylation.

Despite being unable to activate the cognate ghrelin receptor (GHS-R), unacylated ghrelin (UAG) possesses a unique activity spectrum that includes promoting bone marrow adipogenesis. Since a receptor mediating this action has not been identified, we re-appraised the potential interaction of UAG with GHS-R in the regulation of bone marrow adiposity. Surprisingly, the adipogenic effects of intra-bone marrow (ibm)-infused acylated ghrelin (AG) and UAG were abolished in male GHS-R-null mice. Gas chromatography showed that isolated tibial marrow adipocytes contain the medium-chain fatty acids utilised in the acylation of UAG, including octanoic acid. Additionally, immunohistochemistry and immunogold electron microscopy revealed that tibial marrow adipocytes show prominent expression of the UAG-activating enzyme ghrelin O-acyl transferase (GOAT), which is located in the membranes of lipid trafficking vesicles and in the plasma membrane. Finally, the adipogenic effect of ibm-infused UAG was completely abolished in GOAT-KO mice. Thus, the adipogenic action of exogenous UAG in tibial marrow is dependent upon acylation by GOAT and activation of GHS-R. This suggests that UAG is subject to target cell-mediated activation - a novel mechanism for manipulating hormone activity.

Paradoxical leanness in the imprinting-centre deletion mouse model for Prader-Willi syndrome.

Prader-Willi syndrome (PWS), a neurodevelopmental disorder caused by loss of paternal gene expression from 15q11-q13, is characterised by growth retardation, hyperphagia and obesity. However, as single gene mutation mouse models for this condition display an incomplete spectrum of the PWS phenotype, we have characterised the metabolic impairment in a mouse model for 'full' PWS, in which deletion of the imprinting centre (IC) abolishes paternal gene expression from the entire PWS cluster. We show that PWS-ICdel mice displayed postnatal growth retardation, with reduced body weight, hyperghrelinaemia and marked abdominal leanness; proportionate retroperitoneal, epididymal/omental and inguinal white adipose tissue (WAT) weights being reduced by 82%, 84% and 67%, respectively. PWS-ICdel mice also displayed a 48% reduction in proportionate interscapular brown adipose tissue (isBAT) weight with significant 'beiging' of abdominal WAT, and a 2°C increase in interscapular surface body temperature. Maintenance of PWS-ICdel mice under thermoneutral conditions (30°C) suppressed the thermogenic activity in PWS-ICdel males, but failed to elevate the abdominal WAT weight, possibly due to a normalisation of caloric intake. Interestingly, PWS-ICdel mice also showed exaggerated food hoarding behaviour with standard and high-fat diets, but despite becoming hyperphagic when switched to a high-fat diet, PWS-ICdel mice failed to gain weight. This evidence indicates that, unlike humans with PWS, loss of paternal gene expression from the PWS cluster in mice results in abdominal leanness. Although reduced subcutaneous insulation may lead to exaggerated heat loss and thermogenesis, abdominal leanness is likely to arise from a reduced lipid storage capacity rather than increased energy utilisation in BAT.

Metformin Prevents Nigrostriatal Dopamine Degeneration Independent of AMPK Activation in Dopamine Neurons.

Metformin is a widely prescribed drug used to treat type-2 diabetes, although recent studies show it has wide ranging effects to treat other diseases. Animal and retrospective human studies indicate that Metformin treatment is neuroprotective in Parkinson's Disease (PD), although the neuroprotective mechanism is unknown, numerous studies suggest the beneficial effects on glucose homeostasis may be through AMPK activation. In this study we tested whether or not AMPK activation in dopamine neurons was required for the neuroprotective effects of Metformin in PD. We generated transgenic mice in which AMPK activity in dopamine neurons was ablated by removing AMPK beta 1 and beta 2 subunits from dopamine transporter expressing neurons. These AMPK WT and KO mice were then chronically exposed to Metformin in the drinking water then exposed to MPTP, the mouse model of PD. Chronic Metformin treatment significantly attenuated the MPTP-induced loss of Tyrosine Hydroxylase (TH) neuronal number and volume and TH protein concentration in the nigrostriatal pathway. Additionally, Metformin treatment prevented the MPTP-induced elevation of the DOPAC:DA ratio regardless of genotype. Metformin also prevented MPTP induced gliosis in the Substantia Nigra. These neuroprotective actions were independent of genotype and occurred in both AMPK WT and AMPK KO mice. Overall, our studies suggest that Metformin's neuroprotective effects are not due to AMPK activation in dopaminergic neurons and that more research is required to determine how metformin acts to restrict the development of PD.

TNFRSF14 aberrations in follicular lymphoma increase clinically significant allogeneic T-cell responses.

Donor T-cell immune responses can eradicate lymphomas after allogeneic hematopoietic stem cell transplantation (AHSCT), but can also damage healthy tissues resulting in harmful graft-versus-host disease (GVHD). Next-generation sequencing has recently identified many new genetic lesions in follicular lymphoma (FL). One such gene, tumor necrosis factor receptor superfamily 14 (TNFRSF14), abnormal in 40% of FL patients, encodes the herpes virus entry mediator (HVEM) which limits T-cell activation via ligation of the B- and T-lymphocyte attenuator. As lymphoma B cells can act as antigen-presenting cells, we hypothesized that TNFRSF14 aberrations that reduce HVEM expression could alter the capacity of FL B cells to stimulate allogeneic T-cell responses and impact the outcome of AHSCT. In an in vitro model of alloreactivity, human lymphoma B cells with TNFRSF14 aberrations had reduced HVEM expression and greater alloantigen-presenting capacity than wild-type lymphoma B cells. The increased immune-stimulatory capacity of lymphoma B cells with TNFRSF14 aberrations had clinical relevance, associating with higher incidence of acute GVHD in patients undergoing AHSCT. FL patients with TNFRSF14 aberrations may benefit from more aggressive immunosuppression to reduce harmful GVHD after transplantation. Importantly, this study is the first to demonstrate the impact of an acquired genetic lesion on the capacity of tumor cells to stimulate allogeneic T-cell immune responses which may have wider consequences for adoptive immunotherapy strategies.