Leptin and ghrelin: Sewing metabolism onto neurodegeneration

Accepted Manuscript Leptin and ghrelin: Sewing metabolism onto neurodegeneration Paola de Candia, Giuseppe Matarese PII:

S0028-3908(17)30622-6

DOI:

10.1016/j.neuropharm.2017.12.025

Reference:

NP 7002

To appear in:

Neuropharmacology

Received Date: 17 October 2017 Revised Date:

11 December 2017

Accepted Date: 13 December 2017

Please cite this article as: de Candia, P., Matarese, G., Leptin and ghrelin: Sewing metabolism onto neurodegeneration, Neuropharmacology (2018), doi: 10.1016/j.neuropharm.2017.12.025. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

Leptin

and

ghrelin:

sewing

2

neurodegeneration.

3

Paola de Candia1* and Giuseppe Matarese2,3*

metabolism

onto

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Endocrinologia e Oncologia Sperimentale, Consiglio Nazionale delle Ricerche (IEOS-

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CNR), 80131 Naples, Italy; 3Dipartimento di Medicina Molecolare e Biotecnologie

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Mediche, Università di Napoli “Federico II”, 80131 Naples, Italy.

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* To whom correspondence should be addressed: Prof. Giuseppe Matarese,

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Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli

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“Federico

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[email protected] or Dr. Paola de Candia, PhD, Via Fantoli 16/15, 20138

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Milan, Italy, Phone: +39 02 55406577, E-mail: [email protected]

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Italy,

Phone/Fax:

+39-0817464580,

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Napoli,

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II”,

Keywords: neurodegeneration, neuroinflammation, metabolism, leptin, ghrelin

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MultiMedica, 20138 Milan, Italy; 2Laboratorio di Immunologia, Istituto di

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E-mail:

ACCEPTED MANUSCRIPT Abstract:

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Life expectancy has considerably increased over the last decades. The negative

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consequence of this augmented longevity has been a dramatic increase of age-related

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chronic neurodegenerative diseases, such as Alzheimer’s, Parkinson’s and multiple

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sclerosis. Epidemiology is telling us there exists a strong correlation between the

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neuronal loss characterizing these disorders and metabolic dysfunction. This review

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aims at presenting the evidence supporting the existence of a molecular system linking

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metabolism with neurodegeneration, with a specific focus on the role of two hormones

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with a key role in the regulatory cross talk between metabolic imbalance and the

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damage of nervous system: leptin and ghrelin.

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1. Introduction

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The industrialized world population constantly ages with a progressive rise of cancer,

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cardiovascular diseases, metabolic disorders and neurodegenerative conditions (Niccoli

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and Partridge 2012). Modern medicine has succeeded to increase human life span, but

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we now need to develop strategies to suppress/delay aging-dependent pathologies.

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In this context, a critical clinical question is whether these pathological conditions are

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independent or instead interconnected. Long known to be associated with diabetes and

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cardiovascular disease, obesity has been more recently associated with decreased brain

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size, lower density of the gray matter and cognitive function (Dixon 2010, Debette, Wolf

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et al. 2014, Climie, Moran et al. 2015). Furthermore, higher weight represents a risk

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factor for the development of Parkinson’s disease, Alzheimer and vascular dementia

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(Abbott, Ross et al. 2002, Kivipelto, Ngandu et al. 2005, Xu, Atti et al. 2011). Importantly,

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an observational study performed in rhesus macaques succeeded to show that caloric

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restriction can not only lower the incidence of age-related pathologies such as diabetes,

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cancer and cardiovascular diseases, but it is also able to preserve the gray matter

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volume in several subcortical regions, the lateral temporal cortex and frontal lobe,

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critical areas that exert motor and executive functions (Colman, Anderson et al. 2009).

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Therefore, both human studies and animal models concur to suggest a close

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relationship between metabolic imbalance (influenced by life style and diet) and the

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pathological processes leading to neurodegeneration. In this context, we will here

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examine the role played by leptin and ghrelin. These peptides are not mere regulators of

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appetite/feeding but are directly involved in neuronal death induced by diversified

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pathological conditions and play a role in neuroprotection and brain function.

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2. The vicious cycle of defective metabolism, dysregulated

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inflammation and neurodegeneration.

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Neurodegeneration causes the alteration of neuronal structure and function, and

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consequent increased neuron death in the central nervous system (CNS). In this review,

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we will mostly focus on the two most frequent neurodegenerative conditions:

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Alzheimer's and Parkinson's diseases, and the most common cause of neurological

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disability in young adults, multiple sclerosis.

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77 2.1. Alzheimer’s disease.

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Alzheimer's disease (AD) is a progressive illness with an estimated prevalence of 10–

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30% in the population >65 years of age and decades-long preclinical and prodromal

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phases (Masters, Bateman et al. 2015). In AD, neuronal death begins in the temporal

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lobe and then in the parietal lobe causing progressive short and long-term memory loss,

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cognitive impairment and psychiatric disorders (Murray, Kumar et al. 2014, Masters,

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Bateman et al. 2015). In 1906, Alois Alzheimer identified neuritic plaques and

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neurofibrillary tangles as prominent neuropathologic features in post-mortem brains of

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certain patients. Much later, it was discovered that neuritic plaques are formed by the

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abnormal deposition of amyloid β-peptide (Aβ) at the extracellular level, and

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intracellular protein aggregates composed of hyper-phosphorylated tau protein (pTau)

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represent the major components of neurofibrillary tangles (NFTs) (Jeong 2017). Aβ

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plaques in concert with pTau are believed to be the major drivers of AD

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neurodegeneration: Aβ deposition starts causing alternations in learning and memory

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circuitry before clinical AD onset, while aging-associated damage of mitochondria

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collaborates with Aβ and pTau to produce synapse loss and cognitive impairment

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(Jeong 2017).

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In 1817, James Parkinson described a disease that was subsequently named after him as

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the “Shaking Palsy” and at the end of the 1800s it was proposed that the anatomical

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substrate of Parkinson’s disease would be a lesion of the substantia nigra. Nowadays, it

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is well established that the first neurons to degenerate are located the substantia nigra

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pars compacta (SNc), which produces dopamine (DA), central to motor control

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(Bernheimer, Birkmayer et al. 1973, Davie 2008).

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As a consequence, patients suffer from typical motor symptoms such as resting tremor,

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postural instability and rigidity, inability to move muscles voluntarily (akinesia) and

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freezing of gait. They also tend to show non-motor symptoms, including sleep disorders,

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cognitive impairment, apathy, depression and progressive dementia (Antonini, Barone

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et al. 2012, Kim, So et al. 2014). The classical neuro-pathological feature is the

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development of intracytoplasmic aggregates of α-synuclein, termed Lewy bodies.

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Analyzing post-mortem human brain tissue from PD, it was observed that the

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development of Lewy bodies is associated with microtubule regression, mitochondrial

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loss and nuclear degradation in neurons. Lewy bodies are also believed to be directly

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responsible of membrane damage, dysregulation of Ca2+ exchange, oxidative

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impairment and consequent cell death (Spillantini, Schmidt et al. 1997).

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2.3. Multiple Sclerosis.

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Multiple sclerosis (MS) is a chronic inflammatory disease of the CNS that, differently

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from AD and PD, mostly affects younger people (onset ranging from 20 to 50 years)

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ACCEPTED MANUSCRIPT (Milo and Kahana 2010). The course of MS is highly varied and unpredictable. In the

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majority of affected patients, the disease is initially characterized by reversible

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neurological symptoms, which are often followed by progressive neurological

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deterioration over time. The cause of MS is not fully elucidated, but it appears to involve

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both genetic susceptibility and non-genetic triggers, such infections, life style and

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environmental factors, that together result in a self-sustaining autoimmune disorder

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characterized by recurrent immune attacks on the CNS. In particular, MS pathogenic

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target is the myelin sheath that is progressively destroyed by the action of autoreactive

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immune cells. Nonetheless, it is now well recognized that the neurological disability in

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MS patients also directly depends on the progressive axonal loss (Dutta and Trapp

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2007, Stadelmann, Albert et al. 2008, Trapp and Nave 2008).

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2.4. The connection between inflammation and brain degeneration.

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A growing body of observational and experimental data shows that a pivotal factor

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intervening in the onset and progression of neuronal degeneration is the cascade of

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processes collectively termed neuroinflammation (Ransohoff 2016). Many studies

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suggest that normal ageing per se increases inflammatory grade in the brain and

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systemically and contributes to progressive neural deficits. Basal levels of many pro-

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inflammatory cytokines increase in older brains, dramatically affecting the survival and

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the function of neurons, glia, and progenitor cells (Murray and Lynch 1998, Bodles and

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Barger 2004, Joseph, Shukitt-Hale et al. 2005, Nolan, Maher et al. 2005). In particular,

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microglia cells, the brain counterpart of macrophages, are neuroprotective in the young

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brain, but change morphologically and modify their inflammatory profile upon aging-

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induced senescence. The increased level of neuroinflammatory cytokines and

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chemokines released by aged over-activated microglia becomes neurotoxic and takes

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ACCEPTED MANUSCRIPT active part in promoting neurodegeneration. It has also been hypothesized that aging-

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related activation of microglia suppresses neurogenesis, known to be profoundly

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affected by the microenvironment, and thus impairs cognitive function (Luo, Ding et al.

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2010).

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In AD, Aβ and NFT accumulation further induces microglia activation, which augments

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the level of neuroinflammatory mediators, which in turn worsen neurodegenerative

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conditions (Tuppo and Arias 2005). Also in PD, there exists a vicious cycle in which

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aggregates of misfolded α-synuclein directly activates glial and other inflammatory cells

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releasing pro-inflammatory molecules which exacerbate cellular sufferance in the

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substantia nigra (Rocha, de Miranda et al. 2015, Wang, Liu et al. 2015, Grimmig,

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Morganti et al. 2016).

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In conclusion, AD and PD are diseases in which a neurodegenerative-centric view has

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given the way to including inflammation as a crucial pathogenic promoter. MS, on the

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other hand, is a condition that has traditionally been considered an autoimmune disease

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and it has then been re-evaluated in light of the neurodegerative component of the

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pathogenic process. In other words, these three conditions are prototypical for the

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intermingling of neuroinflammation and neurodegeneration.

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2.5. The association between metabolic imbalance and neurodegeneration.

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In recent time, it has become clearer and clearer that there exists a link between the

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metabolic state and the neurological symptoms of neurodegenerative diseases. Several

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observational studies introduced solid evidence suggesting a direct relation between

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diet and changes in the brain structure and activity (Solfrizzi, Custodero et al. 2017).

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Higher body mass index and midlife adiposity increase the risk of later dementia and PD

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development (Abbott, Ross et al. 2002, Whitmer, Gunderson et al. 2005); subjects with

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ACCEPTED MANUSCRIPT type 2 diabetes mellitus are more susceptible to develop AD than healthy individuals

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(Ott, Stolk et al. 1999); and adolescence obesity increases the risk of developing MS

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(Munger, Chitnis et al. 2009, Hedstrom, Olsson et al. 2012, Langer-Gould, Brara et al.

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2013). A high-fat diet directly damages brain function via glucotoxicity, i.e. the toxic

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effects of increased glucose or dysregulated insulin sensitivity (Cai, Cong et al. 2012).

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Moreover, AD transgenic mice fed with a high fat diet on one side develop insulin

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resistance, on the other show more severe Aβ accumulation and worse cognitive deficits

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compared to the same mice fed normally (Ho, Qin et al. 2004). In AD specifically, the

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connection between high levels of glucose and disease progression has also a direct

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molecular basis in the role of glucose in the cleavage of tau (Kim, Backus et al. 2009).

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Notably, T2D and AD diseased mice suffer from similar cognitive impairment, vascular

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defects and augmented Aβ deposition in the brain (Carvalho, Cardoso et al. 2012,

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Carvalho, Machado et al. 2013) and AD neurodegeneration is accompanied by a

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dysfunctional utilization of glucose (Steen, Terry et al. 2005, Schrijvers, Witteman et al.

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2010).

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Other sets of studies performed in PD corroborate the hypothesis that drugs used to

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treat T2D may produce significant neuroprotective effects (Aviles-Olmos, Dickson et al.

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2013, Aviles-Olmos, Limousin et al. 2013). Similarly to AD, in brain areas affected by PD,

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it has been observed an impaired expression of insulin receptors and insulin signaling,

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with altered levels of glucose and glucose tolerance, directly contributing to the

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formation of α-synuclein pathological aggregates and suggesting that a dysfunction of

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the insulin/insulin receptor system may precede the decline of the dopaminergic

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neurons (Sandyk 1993, Moroo, Yamada et al. 1994, Figlewicz, Evans et al. 2003).

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Interestingly, low levels of insulin in T2D rats correlate with a decrease in messenger

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RNA for DA transporter and tyrosine hydroxylase in substantia nigra, suggesting an

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impairment of DA signaling in these animals (Figlewicz, Brot et al. 1996). It has also

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been described that mouse models of T2D treated with a neurotoxin used to mimic PD-

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related

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develop a higher grade of brain inflammation and demonstrate increased accumulation

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of α-synuclein compared to the non-diabetic controls, suggesting that diabetes causes a

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worse dopaminergic cell decline in PD conditions (Wang, Zhai et al. 2014). In addition,

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the decrease of glucose uptake by brain cells can alter the intracellular ratio of ATP and

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ADP, impacting the capacity of dopaminergic neurons to produce and release DA (Levin

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2000, Santiago and Potashkin 2013).

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One striking molecule that seems to be the cornerstone at the crossroad of metabolism,

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inflammation and neurodegeneration is called leptin.

(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,

MPTP)

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neurodegeneration

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3. Leptin: the anorexigenic neuroprotective adipokine with a

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twisting pro-inflammatory role.

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3.1. The critical weight of the “thin” gene.

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Leptin is a 167 amino acid long peptide transcribed by the human ob gene, located on

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chromosome 7 (Zhang, Proenca et al. 1994). Even if leptin orthologs in non-mammalian

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animals are significantly different in terms of primary amino-acidic sequence, its

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function seems to be preserved through the conservation of secondary and tertiary

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structures. Leptin crystal structure has revealed a four-helix bundle characteristic of the

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long-chain helical cytokine family (Zhang, Basinski et al. 1997).

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The crucial role of leptin as “satiety factor” or “fasting hormone” is demonstrated by the

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phenotype of leptin-deficient mice (ob/ob) that eat excessively and become profoundly

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obese (Pelleymounter, Cullen et al. 1995). Similarly, mice with a mutation in the leptin

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ACCEPTED MANUSCRIPT receptor that renders it unable to sense this factor (db/db) develop hyperphagic obesity

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and represent a model of spontaneous type 2 diabetes (Chen, Charlat et al. 1996). Leptin

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is mostly produced and released by the adipose tissue in quantity that is proportional to

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fat mass, and its circulating concentrations decline rapidly in response to reduced body

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fat and food intake, providing a dynamic measure of the size of fat storage and acute

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changes in energy balance (Ahima, Prabakaran et al. 1996). Its primary metabolic

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function is to inhibit food intake and stimulate energy expenditure, via receptor-

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mediated actions in the hypothalamus (Jequier 2002). The leptin-leptin receptor axis

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provides a means for the periphery to communicate the metabolic status of the

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organism to the CNS. The crucial relevance of the leptin signaling in human energy

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homeostasis is revealed by the phenotype of individuals who are homozygous for

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inactivating mutations in leptin or leptin receptor: these people are clearly obese,

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extremely hyperphagic and closely resemble the metabolic imbalance of the obese and

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diabetic mice (Montague, Farooqi et al. 1997, Clement, Vaisse et al. 1998). Furthermore,

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the generalized or partial loss of adipose tissue that characterizes individuals with

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either genetic or acquired lipodystrophy syndromes results in leptin deficiency, that in

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turn leads to hyperphagia and insulin resistance. Notably, leptin replacement has

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become a relevant clinical strategy that results in decreased morbidity for these

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individuals (Garg 2004, Diker-Cohen, Cochran et al. 2015).

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Leptin receptors are found in other brain regions than hypothalamus, among which the

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hippocampus, where leptin may play a role in controlling learning and memory. In

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addition, leptin was shown to regulate bone metabolism, angiogenesis, hematopoiesis

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and sexual reproduction (Bennett, Solar et al. 1996, Chehab, Lim et al. 1996, Sierra-

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Honigmann, Nath et al. 1998, Ducy, Amling et al. 2000). Given its central role in body

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ACCEPTED MANUSCRIPT weight regulation, leptin name comes from the Greek word “leptos” that means “thin”,

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but this hormone has a much more pleiotropic influence than originally believed.

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3.2. The neuroprotective effect of leptin

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Leptin is an important neurotrophic factor, involved in the regulation of neuronal

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development, function and survival. The actual participation of leptin in the

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pathogenesis of neurodegeneration has been extensively studies in both ob/ob and

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db/db mice: these animals show reduced brain volume and a plethora of relevant

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proteins display an aberrant brain expression pattern such as syntaxin-1,

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synaptosomal-associated protein-25, synaptobrevin, myelin basic protein and glial

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fibrillary acidic protein. When the same mice are treated with leptin, the brain weight,

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overall protein content and locomotor activity get normalized, demonstrating that

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leptin indeed controls neuronal and glial maturation and myelin development (Ahima,

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Bjorbaek et al. 1999, Schwartz and Baskin 2013).

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In human, there exists an association between leptin levels in the circulation and

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incidence of dementia, with leptin being a protective parameter (Lieb, Beiser et al. 2009,

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Bigalke, Schreitmuller et al. 2011). Leptin signaling has been extensively analyzed in AD,

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where it has been shown to diminish the level of tau phosphorylation in neuronal cells

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and to inhibit Aβ accumulation via activating AMPK and downregulating the expression

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of the γ-secretase components (Fewlass, Noboa et al. 2004, Greco, Sarkar et al. 2009,

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Greco, Bryan et al. 2010, Niedowicz, Studzinski et al. 2013). While leptin levels were

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found increased in both CSF and hippocampal tissue of AD patients, the level of leptin

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receptor mRNA was instead observed to be decreased in AD brain, with the receptor

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protein trapped in neurofibrillary tangles, suggesting a dramatic impairment of leptin-

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dependent signaling (Bonda, Stone et al. 2014). These observations suggest that leptin

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imbalance can accelerate cognitive decline and AD progression, while AD process, on its

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ACCEPTED MANUSCRIPT part, can block leptin signaling, in a downward loop of progressive neurodegeneration

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and worsening metabolic defects. Clinical trial data demonstrate that leptin

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administration is safe for long term use in humans, that suggests a potential application

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of leptin as a therapy for AD. In mice, chronic administration of leptin to AD-transgenic

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animals also results in better cognitive performance and behavioral improvements, as

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early as after 4 weeks of treatment (Greco, Bryan et al. 2010). Moreover, leptin

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replacement in patients with congenital leptin deficiency leads to a rise in gray matter

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volume (Matochik, London et al. 2005).

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3.3 Leptin central role in enhancing the function of the immune system

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Giving support to the knowledge that leptin exerts pleiotropic effects in periphery, it

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has been observed that ob/ob and db/db mice are not only obese but also show

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significant alterations of the immune system (Lord, Matarese et al. 1998, Lord, Matarese

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et al. 2001). Indeed, several immune cell types are shown to express detectable level of

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the leptin receptor (Procaccini, Jirillo et al. 2012) and leptin is extremely powerful in

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regulating the activity of cells of the native and the adaptive immune system. It

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stimulates neutrophils, regulates phagocytic function of macrophages and the

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cytotoxicity of natural killer cells and activates dendritic cells (Gainsford, Willson et al.

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1996, Loffreda, Yang et al. 1998, Caldefie-Chezet, Poulin et al. 2001, Tian, Sun et al.

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2002, Caldefie-Chezet, Poulin et al. 2003, Macia, Delacre et al. 2006). Leptin also

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modulates the proliferation of CD4+ T helper (Th) cells (Lord, Matarese et al. 1998). In

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particular, it activates the proliferation and differentiation of CD4+ T into pro-

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inflammatory Th-17 cells (Lord, Matarese et al. 1998). To address the cell-intrinsic role

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of leptin signaling in T cells, the leptin receptor was conditionally targeted in these cells

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specifically: the study succeed to show that LepR deficiency causes a selective defect in

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the development of Th17 phenotype and both autoimmune and protective responses

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ACCEPTED MANUSCRIPT (Reis, Lee et al. 2015). At the same time, leptin blocks the activity and growth of the

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anti-inflammatory T regulatory (Treg) cell subset: it has been reported that level of

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circulating leptin and frequency of Treg cells indeed inversely correlate (Wagner,

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Brandhorst et al. 2013).

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Experimental autoimmune encephalomyelitis (EAE) is the most commonly used

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experimental model for the human MS (Constantinescu, Farooqi et al. 2011). When

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ob/ob mice are induced to develop EAE by myelin-specific T cell stimulation, they resist

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to disease induction and progression, showing low levels of IFN-γ and high levels of the

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anti-inflammatory IL-4 (Matarese, Di Giacomo et al. 2001, Matarese, Sanna et al. 2001,

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Sanna, Di Giacomo et al. 2003). Consistently, leptin neutralization in wild type EAE mice

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is also able to delay disease progression, by blocking the action of auto-reactive

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lymphocytes (De Rosa, Procaccini et al. 2006, Galgani, Procaccini et al. 2010). On the

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other hand, leptin replacement in ob/ob mice is sufficient to render them susceptible to

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disease with a strong shift towards a pro-inflammatory cytokine pattern (Matarese, Di

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Giacomo et al. 2001, Matarese, Sanna et al. 2001, Sanna, Di Giacomo et al. 2003); and

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leptin administration to wild type mice increases the severity of EAE (Sanna, Di

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Giacomo et al. 2003). In addition to its pro-inflammatory effect on lymphocytes, leptin is

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believed to potentially worsen blood–brain-barrier dysregulation, because it was

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possible to reduce lymphocyte infiltration into spinal cord by specifically blocking leptin

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signaling in the endothelium (Ouyang, Hsuchou et al. 2014). Notably, MS patients show

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high level of leptin expression in active inflammatory lesions of the CNS and in the sera

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of MS patients before relapses upon treatment with IFN-β (Lock, Hermans et al. 2002,

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Batocchi, Rotondi et al. 2003, Matarese, Carrieri et al. 2005, Olama, Senna et al. 2012,

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Emamgholipour, Eshaghi et al. 2013, Carbone, De Rosa et al. 2014). Specific gene

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expression analysis of T helper lymphocytes from active MS lesions has confirmed the

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ACCEPTED MANUSCRIPT presence of elevated transcriptional level of leptin (Lock, Hermans et al. 2002).

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Environmental metabolic cues can modify the ratio between inflammation and immune

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tolerance by skewing T-cell fate decisions toward either the Th1/Th17 pro-

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inflammatory or Treg immune suppressive cell lineages, because these cell subsets have

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a different response to leptin levels (MacIver, Michalek et al. 2013).

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These studies strongly support the notion that leptin represents the crucial in vivo

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molecular link between metabolic homeostasis and inflammatory dysregulation. Caloric

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restriction has been described to significantly increase the survival and lifespan in EAE

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mice, reducing inflammation, demyelination, and axon damage, to the extent that

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dietary intervention is now considered a potentially efficacious new strategy for MS

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treatment (Piccio, Stark et al. 2008). One of the beneficial effects of fasting may indeed

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depend on reduced circulating leptin levels that in turn has a significant beneficial

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repercussion on the inflammatory state (Liu, Yu et al. 2012). In addition, caloric

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restriction leads to increased blood concentrations of anti-inflammatory corticosterone

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and adiponectin (Piccio, Stark et al. 2008); it also augments the level of ghrelin, another

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pivotal hormone regulating metabolism, but not only (Esquifino, Cano et al. 2004,

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Esquifino, Cano et al. 2007).

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4. Ghrelin: the orexigenic peptide with a consistent

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neuroprotective and anti-inflammatory function.

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4.1 The complex ghrelin system.

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Ghrelin is predominantly produced by specialized endocrine cells of the stomach

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(Kojima, Hosoda et al. 1999), and circulating ghrelin increases before meals to levels

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that stimulate food intake and also increases following food deprivation and after

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ACCEPTED MANUSCRIPT various forms of weight loss; in other words, its rise is expected in conditions of caloric

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restriction (Cummings, Purnell et al. 2001, Nagaya, Uematsu et al. 2001, Otto, Cuntz et

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al. 2001, Cummings 2006). Its metabolic effects are opposite to those of leptin, as it

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stimulates food intake, decreases energy expenditure and regulates growth hormone

344

secretion, glucose-sensing and glucose homeostasis (Cummings and Foster 2003,

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Nogueiras, Tschop et al. 2008). Ghrelin also influences body weight by promoting food-

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reward behaviors and increasing the mRNA expression of fat storage–promoting

347

enzymes in the adipose tissue (Perello, Sakata et al. 2010). Diet-induced obesity was

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shown to dramatically dysregulate ghrelin function in food intake and metabolic control

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(Briggs, Enriori et al. 2010).

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The mature form of ghrelin, a 28 amino acid long peptide, results from the proteolytic

351

processing of a precursor peptide named preproghrelin. In humans, the peptide is

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encoded by a single-copy gene (GHRL) located on the short arm of chromosome 3, that

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shows high regulatory complexity due to alternative sites for transcription initiation

354

and alternative splicing mRNA variants due to exon skipping or intron retention (Seim,

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Herington et al. 2009, Sato, Nakamura et al. 2012). In particular, a variant that lacks the

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coding region of exon 3 (Ex3-deleted ghrelin), and another variant generated by

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retention of intron 1 (In1-ghrelin) are particularly interesting because their

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conservation in other mammalian species and/or overexpression in cancer samples

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suggest that these variants might exert important patho-physiological role, yet to be

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identified (Yeh, Jeffery et al. 2005, Gahete, Cordoba-Chacon et al. 2011).

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In addition, the final steps of preproghrelin proteolytic processing generate both the

362

mature native ghrelin peptide, and an additional C-terminal peptide named C-ghrelin,

363

whose further proteolytic process generates a functional peptide, obestatin,

364

hypothesized to be the antagonist hormone for ghrelin (Zhang, Ren et al. 2005, Sato,

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ACCEPTED MANUSCRIPT Nakamura et al. 2012). To add further complexity to the ghrelin system, native ghrelin

366

peptide can be modified through acylation (addition of an octanoyl group) of the highly

367

conserved third serine residue: according to the acylation status, the peptide is named

368

unacylated ghrelin (UAG) or acylated-ghrelin (AG) (Kojima, Hosoda et al. 1999, Nishi,

369

Yoh et al. 2011). The acylation process is mediated by an enzyme known as ghrelin-O-

370

acyl-transferase or GOAT, believed to act before the proteolytic processing (Garg 2007).

371

Notably, while UAG represents the large majority of total blood ghrelin (Patterson,

372

Murphy et al. 2005), it is unable to bind the classic ghrelin receptor (GHSR1a), leaving

373

its potential biological role not yet elucidated. It is also unknown whether UAG is the

374

product of the incomplete acylation of the nascent ghrelin peptide, or is also the product

375

of mature AG de-acylation (Kojima, Hosoda et al. 1999, Nishi, Yoh et al. 2011).

376

4.2 Ghrelin: not just a question of hunger.

377

In addition to mediating opposite effects in the neuro-hypothalamic axis, ghrelin also

378

displays reverse action relative to leptin on immune activity in EAE.

379

administration has been shown to significantly associate with a reduction of pro-

380

inflammatory cytokine expression (TNF-alpha, IL-1β, and IL-6) and diminished cellular

381

infiltrates in the spinal cord (Theil, Miyake et al. 2009). The anti-inflammatory role of

382

ghrelin is striking to the point that it has projected this hormone in the arena as a

383

potential strategy for the management of MS.

384

Ghrelin receptors are expressed in a widespread manner in the CNS, suggesting that this

385

hormone is not only involved in metabolism and inflammation, but plays other essential

386

brain functions, such as learning and memory, anxiety control, reward, mood, and sleep

387

(Muller, Nogueiras et al. 2015). Several beneficial effects of ghrelin on neurons have

388

been described: it protects mitochondria, inhibiting reactive oxygen species formation

389

and cytochrome-c release; it regulates the level of intracellular calcium and nitric oxide,

Ghrelin

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ACCEPTED MANUSCRIPT which has a direct effect on memory function; similarly to leptin, ghrelin also protects

391

neurons from the amyloid-associated toxicity (Chung, Kim et al. 2007, Moon, Choi et al.

392

2011, Gomes, Martins et al. 2014). Ghrelin levels change with ageing and a diminished

393

ghrelin signaling has been shown to precede impairment in cognitive testing and its

394

serum level was found to inversely associate with cognitive function in non-demented

395

elderly people (Rigamonti, Pincelli et al. 2002, Spitznagel, Benitez et al. 2010). A very

396

recent report shows that ghrelin is able to attenuate diabetic-related rat

397

encephalopathy, having a beneficial effect on both neuroinflammation and cognitive

398

impairment. In particular, ghrelin was found to ameliorate astrocytic activation, reduce

399

pro-inflammatory factors such as interleukin-6 and tumor necrosis factor-α and reverse

400

down-regulation of mature nerve growth factor (NGF) in experimental diabetic rats

401

(Zhao, Shen et al. 2017). Ghrelin was also shown to prevent neuronal degeneration and

402

improve motor coordination in a mouse traumatic brain injury model by blocking

403

apoptosis and blood-brain barrier dysfunction (Lopez, Lindsay et al. 2014).

404

Ghrelin dysregulation has been suggested to contribute to the severe cognitive deficit

405

observed in AD, since its expression was found strikingly reduced in one of the cortical

406

regions most affected in AD, the temporal gyrus, when comparing AD to healthy

407

subjects (Gahete, Rubio et al. 2010). Ghrelin level is also diminished in PD, with PD

408

patients showing lower plasma ghrelin concentrations compared to healthy individuals,

409

that demonstrates an impaired ghrelin production in PD conditions (Fiszer,

410

Michalowska et al. 2010). Furthermore, ghrelin production is reduced in pre-motor

411

stage of PD, suggesting a direct implication of this hormone in disease progression

412

(Andrews, Erion et al. 2009), while ghrelin administration is able to protect DA neurons

413

from MPTP-induced death, reduce microglial activation and the levels of TNF-α, IL-1β,

414

and NO (Moon, Kim et al. 2009).

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ACCEPTED MANUSCRIPT Independently of the presence of the acyl group, ghrelin is able to protect primary

416

cultured rat cortical neurons from ischemic injury and glucose deprivation, by

417

increasing phosphorylation of extracellular signal-regulated kinase (ERK)1/2, Akt,

418

glycogen synthase kinase-3β (GSK-3β), Bcl-2/Bax ratio, and β-catenin nuclear

419

translocation and preventing cytochrome c release and caspase-3 activation (Chung,

420

Seo et al. 2008). These results suggest the involvement of a receptor that is distinct from

421

GHS-R1a. Intriguingly, UAG, but not AG, would exert this action by binding and blocking

422

the action of CD36, a scavenger receptor able to recruit and activate microglia and

423

secretion of inflammatory mediators and cause neuronal dysfunction and death

424

(Bulgarelli, Tamiazzo et al. 2009). On the other hand, another study identifies acylated

425

ghrelin as the form responsible for in vivo neuroprotection in a mouse model of PD:

426

while administration of acylated ghrelin in ghrelin KO mice can attenuate the MPTP-

427

induced neuronal loss and reduce glial activation in the substancia nigra, administration

428

of des-acylated ghrelin to the same mice fails to do the same, suggesting that

429

pharmacological approaches impacting the ratio between acyl ghrelin to des-acyl

430

ghrelin may have clinical efficacy to slow PD progression (Bayliss, Lemus et al. 2016).

431

Notably, the dopaminergic receptor D1R and the ghrelin receptor GHSR1a were

432

demonstrated to bind and form heterodimers in vitro and described to co-localize in

433

various areas of the brain (Jiang, Betancourt et al. 2006), and it was subsequently

434

reported that GHSR1a receptor can modify dopaminergic signaling by enhancing DA

435

release and thus reducing PD motor symptoms (Kern, Albarran-Zeckler et al. 2012).

436

It has been observed that the neuroprotective effect of caloric restriction in a mouse

437

experimental model of PD depends on the function of ghrelin, in particular on its

438

capacity to activate AMPK in dopamine neurons (Bayliss, Lemus et al. 2016). AMPK

439

senses cellular energy, increases adenosine triphosphate (ATP) production and

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ACCEPTED MANUSCRIPT suppresses energy consumption in conditions of cell stress (Hardie, Ross et al. 2012).

441

Through AMPK, ghrelin is known to activate peroxisome proliferator-activated receptor

442

γ coactivator-1α (PGC-1α) (Canto and Auwerx 2009), a master regulator of

443

mitochondrial biogenesis, known to be dysfunctional during PD progression and

444

pinpointed to be significantly decreased in DA neurons from PD patients (Zheng, Liao et

445

al. 2010). The positive effect of ghrelin on PGC-1 determines enhanced mitochondrial

446

health and may reduce dopaminergic cell loss. The relevance of this ghrelin target in

447

preventing neurodegeneration is demonstrated by the phenotype of PGC-1α null mice

448

that result to be more susceptible to MPTP-induced PD (St-Pierre, Drori et al. 2006). On

449

the other hand, ghrelin target AMPK is central in the induction of autophagy, the

450

process

451

removed/destroyed/digested. When the energy balance is low, AMPK activation leads

452

to mTOR inhibition and subsequent autophagy induction.

453

4.2 mTOR and autophagy

454

The mechanistic target of rapamycin (mTOR) protein is a highly conserved serine–

455

threonine kinase that senses the energy status and regulates anabolism, cell-cycle

456

progression, growth and autophagy. Two different complexes of mTOR (mTORC1 and

457

mTORC2) show different sensitivity to rapamycin and associate with different protein

458

regulatory partners. The nutrient-sensitive complex mTORC1 is central for the

459

induction of protein synthesis through phosphorylation of ribosomal S6 kinase (S6K).

460

In recent times, the crucial role of mTORC1 as signaling target of both leptin and ghrelin

461

is being unveiled. In rodent hypothalamus, leptin has been shown to increase mTORC1

462

activity, and mTOR signaling to mediate the effect of leptin in food intake regulation.

463

Either mTORC1 inhibition by rapamycin, or the deletion of mTOR target S6K is able to

damaged

or

unnecessary

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components

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ACCEPTED MANUSCRIPT disrupt leptin's anorectic effect, demonstrating that this intracellular signaling pathway

465

is required for the appetite-suppressing effects of this hormone (Cota, Proulx et al.

466

2006, Cota, Matter et al. 2008). Ghrelin has also been shown to promote

467

phosphorylation of S6K1 in the rat hypothalamus, an effect that is suppressed by

468

rapamycin, suggesting that ghrelin role in food intake, adiposity and insulin secretion is

469

actually controlled by the mTORC1/S6K1 pathway in the central nervous system

470

(Stevanovic, Trajkovic et al. 2013). Since ghrelin and leptin play opposite roles in food

471

intake, it has been hypothesized that mTORC1 is a switching balance that mediates the

472

distinct effects of these two peptides in metabolic regulation, depending on nutritional

473

status and neuronal activity (Haissaguerre, Saucisse et al. 2014).

474

Interestingly, a systemic dysregulation of the mTOR signaling cascade is present in the

475

brain but also in peripheral lymphocytes of Alzheimer's disease patients before the

476

onset of the disease, suggesting this specific pathway may represent a risk factor for

477

Alzheimer (Yates, Zafar et al. 2013). Indeed, mTOR signaling pathway has been shown

478

to be involved in the balance of phosphorylated/non-phosphorylated tau, and its

479

localization and aggregation, suggesting an mTOR direct effect in tau deposition (Kang

480

and Cho 2015, Tang, Ioja et al. 2015). In neuronal cell lines and primary neurons, PD

481

inducers such as 6-hydroxydopamine or N-methyl-4-phenylpyridine reduce cell

482

viability in parallel with a suppression of mTOR function and activation of caspase-3

483

and poly-ADP ribose polymerase (PARP) cleavage. Furthermore, overexpression of

484

mTOR reduced cell death in response to the PD toxins. It was also found that the

485

suppression of Akt and the activation of AMPK were important contributors of neuronal

486

cell death (Xu, Liu et al. 2014). Another report has shown that mTOR activation can

487

prevent oxidative stress-mediated cell death, while treatment with rapamycin

488

potentiated this type of cell death in dopamine neurons. Importantly, a crucial

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ACCEPTED MANUSCRIPT mechanism involved in the capacity of mTOR to protect the neurons was found to be

490

autophagy, mTOR being the key regulator of the autophagic process in response to

491

growth factors and cell stress (Saxton and Sabatini 2017). Recent work suggests that

492

dysregulated autophagy is implicated in different neurodegenerative diseases with the

493

common denominator of cellular toxicity associated with misfolded protein

494

accumulation. The formation of α-synuclein aggregates in PD is inhibited by the

495

induction of autophagy, leading to the hypothesis that the correction of autophagic

496

process may rescue from disease progression (Mishra, ur Rasheed et al. 2015).

497

Consistently, treatment with an autophagy inhibitor is able to protect the dopamine

498

cells (Choi, Kim et al. 2010). Other studies, though, tell a different story. Rapamycin has

499

been shown to protect against neuron death in in vivo models of PD and the

500

downregulation of mTOR signaling pathway and the consequent recovery of

501

macroautophagy secondary to curcumin treatment has a protective effect against the

502

neurodegenerative pathology in a missense mutation of α-synuclein model of

503

hereditary PD (Malagelada, Jin et al. 2010, Jiang, Zhang et al. 2013).

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5. Conclusion and Perspectives

506

Animal models and human studies strongly suggest that there is a close interconnection

507

between metabolism, inflammation and neurodegeneration. Several observations

508

indicate that therapies targeting systemic inflammation may provide benefit for those

509

afflicted by neurodegenerative disorders; and that the two conditions of

510

neurodegenerative disorders and metabolic dysregulation share specific molecular

511

mechanisms to the point that agents with proven efficacy in one may be useful against

512

the other. In other words, therapies aimed at re-establishing metabolic homeostasis

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ACCEPTED MANUSCRIPT may have also an efficacy in contrasting cognitive decline. In this context, hormones like

514

leptin or ghrelin, originally believed to be primarily regulators of feeding and appetite,

515

have been later demonstrated to play important roles in brain function,

516

neuroprotection and neuroinflammation, linking metabolic regulation with brain health

517

and disease (Figure 1).

518

It is important to point out that leptin and ghrelin have been discovered to play a role in

519

CNS pathologies other than neurodegeneration. By regulating the hypothalamic-

520

pituitary-adrenal axis, ghrelin affects anxiety and mood disorders, depression, fear and

521

alcohol dependence (Leggio 2010, Spencer, Emmerzaal et al. 2015, Zarouna, Wozniak et

522

al. 2015). At the same time, many human studies have associated leptin levels with

523

depression and anxiety, sleep disturbances, behavioral disorders, social isolation and

524

even suicide attempts (Westling, Ahren et al. 2004, Atmaca, Tezcan et al. 2005, Lu, Kim

525

et al. 2006, Lawson, Miller et al. 2012, Zarouna, Wozniak et al. 2015). Still, the role of

526

ghrelin and leptin in mood regulation has not been yet clearly dissected, and further

527

studies are needed to reveal potential common pattern of emotional changes and

528

level/biological impact of these two hormones. All these complex and pleiotropic effects

529

of leptin and ghrelin on CNS and the periphery must be taken into account in future

530

clinical trials evaluating the beneficial and adverse consequences of their

531

administration in humans with the final goal of identifying novel therapeutic

532

approaches that, combined with lifestyle modifications, can target neuroinflammation

533

and synergistically combat neurodegeneration.

534

Moreover, we cannot ignore that, at the systemic level, leptin and ghrelin act in concert

535

with a plethora of other signals. Beside leptin, many different molecules differently

536

impact metabolism and immunological functions, such as adipose-derived adipokines

537

resistin, angiopoietin-like protein 2, adiponectin, visfatin and adipsin (Bluher and

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ACCEPTED MANUSCRIPT Mantzoros 2015). In particular, the concomitant influence of leptin on eating and

539

energy expenditure is believed to work in concert with the short-term effects induced

540

by amylin, a pancreatic β-cell hormone co-released with insulin in response to food

541

intake. Importantly amylin precursor is also expressed by hypothalamic neurons and

542

positively regulated by leptin; amylin blockade decreases the effects of leptin on

543

neurons in vivo and in vitro, suggesting hypothalamic amylin acts in concert with leptin

544

(Li, Kelly et al. 2015). Regarding the biological role of ghrelin, we need to acquire more

545

knowledge on the overlapping or distinct effect of the acylated, un-acylated and de-

546

acylated forms of ghrelin, but also on the contribution of the multiple different mRNA

547

variants. Beside the above-mentioned Ex3-deleted ghrelin and In1-ghrelin, some of the

548

splicing variants generate peptides with small changes in their sequences compared

549

with native ghrelin, such as des-Gln14-ghrelin, which is identical to native ghrelin

550

except for the deletion of one glutamine residue (Gln14), whose difference in function is

551

still totally obscure (Sato, Nakamura et al. 2012).

552

Finally, a fuller understanding of the grid composed by the myriad of these molecules

553

and their reciprocal interaction, and a better knowledge of the effect of leptin and

554

ghrelin on intracellular central regulatory hubs, such as mTOR, may provide not only

555

new approaches to improve these two hormones signaling and sensitivity, but also a

556

more in-depth comprehension of the strings that link metabolism and brain health.

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Acknowledgements

562

This work was supported by funds from the European Foundation for the Study of

563

Diabetes/Juvenile Diabetes Research Foundation/Lilly program 2015. PdC is also

564

supported by the National Multiple Sclerosis Society NMSS (PP-1606-24687) and

565

Fondazione Italiana Sclerosi Multipla FISM (2016/R/10). GM is supported by the Italian

566

Space Agency (ASI, 2014-033-R.O) and Fondazione Italiana Sclerosi Multipla FISM

567

(2016/R/18).

568

The authors declare that they have no conflicts of interest with the contents of this

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article.

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Ghrelin

Lep n

Health

Pro-inflammatory: - Ac vates pro-inflammatory T cells - Reduces Treg func on - Worsens blood-brain barrier dysregula on

Balanced immune/metabolic homeostasis allows: - The neuroprotec ve role of lep n and ghrelin; - The proper control of mTOR pathway and autophagy.

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Disease

Altered control of metabolic func ons and chronic inflamma on: - Impairs the neuroprotec ve role of lep n and ghrelin; - Dysregulates mTOR signaling and leads to defec ve autophagy.

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An -inflammatory: - Reduces microglial ac va on and the pro-inflammatory cytokines Diminishes spinal cord cell infiltrates and auto-immune severity in EAE

Neuroprotec ve: - Controls brain size and a correct protein expression pa ern - Reduces the forma on of Aβ and NFT - Promotes survival of dopaminergic neurons

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Neuroprotec ve: - Protects dopaminergic neurons - Reduces ROS produc on and cytochrome c release - Ameliorates blood-brain barrier dysfunc on - Improves motor coordina on

Figure 1. The multiple roles played by ghrelin and leptin in the crosstalk between metabolic

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imbalance, neuroinflammation and neurodegeneration. In healthy conditions, regulated levels of

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leptin and ghrelin exert a beneficial effect (green) on the central nervous system. When metabolic balance

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is disrupted, their neuroprotective role is lost and high leptin levels aggravate neuroinflammation and

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consequent neurodegeneration (red).

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HIGHLIGHTS 1. Leptin exerts a neuroprotective role by blocking the formation of Aß and NFT and promoting survival of dopaminergic neurons.

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2. Leptin, on the other hand, also plays a pro-inflammatory role by activating T cells and worsening blood-brain barrier dysregulation.

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3. Ghrelin exerts a beneficial role by protecting dopaminergic neurons, improving motor coordination and ameliorating blood-brain barrier dysfunction.

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4. Ghrelin also plays an anti-inflammatory role by reducing microglial activation and pro-inflammatory cytokines.

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5. The altered control of metabolic functions impairs the neuroprotective role of leptin and ghrelin; and worsens the pro-inflammatory role of leptin.