Vol.5, no. l/2,35-52 (2016) EUROPEAN JOURNAL OF NEURODEGENERATIVE DISEASES

OLD AND NEW EVIDENCE FOR INVOLVEMEI\T IN ALZHEIMER'S DISEASE

ANd R. POSSENTI3 C. SEVER[N{II.2, C. PETRE,LLAI.2,P. PETROCCHI PASSERII tlnstitute of Cett Biology and Neurobiology, CNR, Rome, Italy \IRCCS Fondazione Santa Lucia, Rome, Italy 3Department of Medicine of Systems, UniversiQ of Rome Tbr Vergata, Rome, ItalY

characterized by degeneration of Alzheimer,s disease (AD) is an irreversible degenerative disorder neuronsindifferentbrsinareasandbyprogressivecognitiveandfunctionaldec|ine.variousderanged neuronal death' the inevitable event occurring mectranisms ptay a role in the disease procesi all inaucing inAD.Nove|therapeuticapproachesusingtlisease.motlifyingtreatmentarebeinginvestigatedwith of their putative roles as the intention of influencing multiple patrrJways involved in AD. Because the central nervous system, neurotransmitter., n"u"oaodol"toi., uoa n"o"L.agulrtors in havebeentheobjectofconsid€rableresearch.Postmortemstudieshaveprovidedevidencethatsevera| neuropeptide-containingneuronsarepathologicallyalteredinbrainareasofADpatients'aswellas of neuropeptides have been found in in the brain of animal models of AD. in adaition, altered levels cerebrospina|fluid(csF)ofADpatients,gettinginsightsintothepotsntialroleofneuropeptidesinthe biomarkers of this patho|ogy. The patr,opr,y,iorogy ofAD and omering ttre possiritity to itlentify novel since they are generatly neuroprotective and role exerted by neuropeptides seemJparccutarty interesting process€s' The present review widely distributed in brain areas responsible ior learning and memory surnmarizestherecentfindingsonneuropeptideinvolvementinAD,withafocusonthecontribution and chromogranin/secretogranin of thyrotrophin-releasing hormone, ctrolecystokinin, family,describingbrainilistributionandthero|ep|ayedinADandincognitivefunctions,aswe|las provided for the protective role of their neuroprote"U"u p"opa"ti"r. Convincing evidence has been both in vitto 8'nd in rivo, identifying these neuropeptides against n.oroa"gene""tion observed in AD, neuropeptide receptors as potential therapeutic targets' Alzheimer'sdisease(AD)isthemostcommonHistopathologically,thehallmarksofADare neurodegenerativedisease,atTectingmorethantheubiquitouspresenceofintra-neuronalfibrillary extracellular deposits of beta amyloid 20 million individuals worldwide. Iì is clinically tangles and in phvsiological aeieat ana cognitive (AB) fibrils in senile piaques. while characterized by memory proteln conditions the phosphorylation-modified tau impairment, accompanied by neuronal loss-in the microtubules in the central uìJ forebrain, locus stabilizes the axonal cerebral cortex, hippo"a-p,,,, undergo nervous system(CNS)' inAD tauproteinmay coeruleus and dorsal raphe and by a significant phoshorylation' hyperphosphorylation and of basal forebrain n"ioon' in abnormal damage "r'orit'"'gi" brain(PókóskiM,KólmónJ'2008).someothermodifications(nikation'ubiquitination,

chronngt'anin/sect"etogranins Kett words; Alzheimeris disease, TRH, CCK, bradt',

Corresponding aulhot" 2219-585s (20 1 6) Cinzia Severini. Colryright it' b! ÍllOLlf E. s'a s' and Neurobiology' CNR' lrlay tl.' bc ttrrtltcr' CNR.lnstitute of CellBiology Tlis publication and/or article is for incli.,icl'al ttst' onl1' and Via Fosso di Fiorarlo. 64, reprtl

a truncation, shift, prolyl isomerization), leading to Neuropeptides are small proteic molecules (from intraneuronal amassing of tau protein bringing about 3 to 100 amino acids) which mediate or modulate the a disruption of neuronal cell communication (Zhao neuronal communication by binding to specific cell et al, 2014). surface receptors: they can act as true neurotransmitters The amyloid (or Ap) hypothesis (Hardy and or as neuromodulators (Hallberg, 2015). Higgins, 1992) has become the dominant model Radioimmunoassay and immunohistochemistry of AD pathogenesis, indicating a crucial role for have allowed to draw exact distribution maps of the production of Ap which aggregare individual neuropeptides and their receptors in into oligomers and further deposit plaques, as and the central (CNS) and feripheral (pNS) neryous guiding the development ofpotential treatments. The systems. Generally, neuropeptides originate in increased AB production leads to an amyloid core the body of the nerve cell from precursors with formation around which neurites, astrocytes, and high molecular weight (pre-pro-peptides), usually glial cells accumulate, thus forming senile plaques biologically inactive, whose processing leads to the and activating an immune system response (Mrak formation of one or more neuropeptides endowed and Griffin, 2001; Tuppo and Arias, 2005). with biological effects (Hallberg and Nyberg, According to this hypothe sis, soluble AB oligomers 2003). Once released, neuropeptides can function are identified as the principal neurotoxic agent in as neuromodulators reaching their receptors at a AD pathology (Haass Selkoe, & 2007). This so_ considerable distance from the site ofrerease. For this called "amyloid hypothesis', was partially modified special form of endocrine transmission the concept by Selkoe and Hardy (2016), indicating the crucial has been proposed of volume transmission (VT) role exerted by neuritic alteration in the immediate and extra-synaptic neurotransmission (Fuxe et al, vicinity of AD plaques, raising the possibility of 2012),further confirmed by the frequent discrepancy decreased efficiency neurotransmission of along observed between neuropeptides and their cognate them. receptor distribution in many brain areas. The pathophysiology ofAD involves disturbances This transmission mode expands the classical and imbalances occurring in a variefy ofmechanisms. concept of synaptic transmission based on a Besides Ap production, neurofibrillary tangle communication point by point and emphasizes the accumulation and inflammation, various deranged capacity of the transmitter molecules, especially mechanisms, such as chronic oxidative stress, peptides, to influence large target areas. mitochondrial dysfunction, hormone imbalance, unlike classical neurotransmitters, inactivated by mitotic dysfunction, calcium mishandling, and specific synaptic reuptake mechanisms, neuropeptide genetic components play a role in the disease activity is disabled by enzymatic proteolysis process, all inducing neuronal death, the inevitable mediated by various extracellular peptidases, or is event occurring in AD (Anand et al, 2014). The induced by the decrease of their concentration due to disease has been extensively studied looking for a dilution. This neuropeptide degradation can lead to therapy, however acetylcholinesterase inhibitors and the formation of fragments that have similar or very memantine are the only drugs currently approved different biological activities in respect to parent for treatment, nevertheless providing symptomatic peptides (Nyberg and Hallberg, 2007). treatment without altering the course of the disease. Considering the large distribution of As a consequence, it is of great importance to develop neuropeptides in the CNS and their ability ro disease modif ing substances that might counteract modulate cognitive functions, it is not surprising that or slow down neurodegeneration. numerous neuropeptides are differentially affected The role exerted by neuropeptides seems in AD (Table I). particularly interesting since they generally are The role exerted by neuropeptides in AD has neuroprotective and are involved in learnins and been recently reviewed. Van Dam et &1, (2013) memory processes. summarized pathophysiological mechanisms and European Journal of Neurodegenerative Diseases 37

Table l. Changes in neuropeptide levels in AD patients ond AD animal models, compored to the corresponding controls (healthy patients or wild type animals). AD human brain AD human CSF AD animal models

Pep tide Levels References Levels References References

Biggins at a1.,1983 (amigdala) Pekary at al.,l99l TRH j Luo at a1.,2002 (hippocampus) î (rRHGly) Yong-Hong at a\.,2013 (blood) Perry at al., IgUl (cortex) Sagar at al.,1984 Diez at a1.,2003 (hippocampus) J (hippocampus of Mazurek and Beal, 1991 t APP23 mice) (cortex) Lofberg at a1.,1996 lcnrtev't CCK

Struble at al.,1987 (amyloid Drez at a1.,2003 plaques) (hippocampus, cortex î Perry at aL.,1981 (amyloid î and amyloid plaques plaques) of V717F mice)

Iores-Marcal at al., 2006 (BK fragment BK î in CSF ofAp infused rats) Blennow et al 1995; Eder (amyloid plaques): et al 1998; Simonsen Marksteiner at al., 2002, et aL2007; Penin et al cgA î Rangon at a|.,2003, v 20ll; Jahn et al2}Ll;

Lechner at a1.,2004, Mattson et al2012: ,i Willis et al20ll Paterson et al2014; Wildsmith at al.2014 Marksteiner et al 2002 Eder et al 1998; Mattson (hippocampus) I et aL2010,2012,2013 Lechner at al. 2004 (cortex) cgB (amyloid plaques): Marksteiner et al 2002: î Lechner at aL.2004: Willis at a|.2008" 2011 AD human brain AD human CSF AD animal models Levels References Levels Willis at a1.,2008 Marksteiner et al 2002 Eder et al 1998; Matsson (amyloid plaques of (hippocampus) et al 2010; Spellman ar î VTl7land K670M/ Lechner at al. 2004 (cortex) al.2015 N67lL mice) SgII Kaufmann 1998 (amyloid plaques, hippocampus, cortex) î Marksteiner et al 2002 (amyloid plaques) Lechner at a\.2004 (amyloid plaques) C. SEVERINI ETAL. 38

SgIII î PIà at al.2013 (cortex)

Winsky-Sommerer at al., 2003 Helwig et al2013 (amyloid plaques of î (cortex) Helwig et al2013 î 7B.2 (amyloid plaques) APP/PSENI mice) Wada et al2004 Hoshino ot al,2014 (brain of APdE9 PToSAAS î Hoshino at al.2014 (amYloid Jahn at al.20ll î plaques) mice) Carrette el al 2003; Jahn at al.20ll; Wujte at al. Cocco at a|.2010 (cortex) ù 2012; Spellman at al. VGF 2015; Hottla at ol.2015; Hendricson at al.2015

different nerve cell therapeutic opportunities of vasopressin and oxytocin, with other neurotransmitters in (Hrabovszky somatostatin, (NPY), corticotropin types in both CNS and peripheral tissues releasing hormone (CRH), , , and Lipositis, 2008). PToTRH mRNA and TRH in vasoactive intestinal peptide (VIP), ' itself are widely distributed throughout the brain opioid peptides, and substance P extra-hypothalamic regions, including the olfactory the (SP). Willis et al, (2011) and Severini et aL, (2016) system, reticular thalamic nucleus, amigdala, where it reviewed the involvement of chromogranins and SP hippócampus, piriform cortex, and striatum, Reichlin, in AD, respectively. plays a neuromodulatory role (Jackson and The aim of the present review is to update 1974; Pekary, 1998) (Fig. 1). information on the activity of old and new TRH has been shown to produce a variety of neuropeptides involved in AD, i.e. thyrotrophin- behavioral changes and neuropharmacological releasing hormone, , bradykinin and effects independent of its thyrotrophin-releasing chromogranin/secretogranin family. properties, by acting via interaction with two different membrane receptors. TRH receptor 1 THYROTROPIN-RELEAS ING HORMONE (TRH-Rl) and receptor 2 (TRH-R2) are typical G-protein-coupled receptors, coupling to Gq ang proteins that Thyrotrophin-Releasing Hormone (TRH) was the G11, pertussin-toxin-insensitive G Osman, 1996)' first hypothalamic releasing hormone to be isolated activate PLC-p (Gershengorn and functions, THR-RI and charactertzed. The name TRH derives from its Consistent with the endocrine nuclei, however, action on the anterior pituitary, where it stimulates, predominates in hypothalamic regions and spinal cord ín vivo and in vitro,not only the synthesis and release is present in brainstem of thyrotrophin (TSH), but also of (PRL), motoneurons where it is involved in autonomic and widely and in some species also of (GH) somatomotor control. THR-R2 mRNA is (Joseph-Bravo et al, 2015). TRH is a tripeptide, distributed throughout the brain with highest levels in identified in 1969, derived from a 242-"amtno the thalamus, cerebral and cerebellar cortex, medial precursor acid protein (ProTRH). In addition to its habenulae, medial geniculate nucleus, pontine neuroendocrine function, stimulating the thyroid nuclei, and the entire reticular formation (Heuer et gland, TRH has functions of neurotransmitter and al, 2000). TRH has been proposed to play a role or neuromodulator in both the CNS and PNS. It was in AD. Significant differences in hypothalamic et al, demonstrated to co-localize and to be co-secreted pituitary functions were observed by Albert European Journal of Neurodegenerative Diseases 39

TRH pyroclu-H is-Pro-N Hz 25s^"W

ccK As p-Tyr-M et-G ly-Trp-M et-Asp-ph e-N H 2

rze aa f .-lW

BK Arg-P ro-P ro-G ly-P h e-Ser-p ro-p h e-Arg-O H

427

Fig' 1' TRH, CCK and BK precursor sequence and brain distribution. The scheme represents the approxímate length and the number of amino ocids of the precursor of TRH, CCK and BK and their amino acid sequence. The leader signal .fbr secretory protein is indicated qs tight shadowed block. The black blocks indicate the biological active fragments identffied' In the right panels are shown images of in situ hybridization for the corcesponding g"i, in mouse CNS (Source from Allen Brain Atlas, mouse. brain-map.org). "*pr"rsion

(1993) between patients with senile dementia of AD functions, suggested a therapeutic potential of type and control subjects undergoing the TRH test, TRH in AD. It was, in fact, demonstrated that TRH while other studies failed to demonstratp a significant administration can improve memory function in AD impairment under TRH stimulation (Gome z et al, patients (Yarbrough and pomara, 19g5; Mellow et 2000). Other works, focusing on the TRH-TSH- al, 1989) and also in the fimbria-fornix lesioned rat thyroid axis, confirrned an abnormal function of model (Bennet et al, 1997). hypothalamic or pituitary functions in AD patients Possible mechanisms for TRH neuroprotection showing that, compared to healthy controls, the AD have been proposed. Among others, it has been patients had significantly lower levels of TRH in the suggested that a signal transduction pathway linking blood (Yong-Hong et aI,2013). TRH with GSK3B activity and tau phosphorylation During the last decades, the relationship between could be responsible for the formation of TRH and AD in human subjects has been extensivery neurofibrillary tangles associated with dementia of studied. Increased levels of TRH were found in AD. By binding to its receptor, mostly the TRH-R2 CSF (Pekary et al, 1991), and decreased levels in subunit receptor, TRH should be able to activate hippocampal regions (Luo et al, 2002) and amigdala the G-protein coupled receptor (GPCR), finally (Biggins et à1, 1983), despite other studies not triggering the MAPK signal pathway to inhibit describing significant differences (yates et al, 19g3; GSK3p activity and prevent tau phosphorylation Bouras et al, 1986;Nemeroff et al, 1989; Banky etaI, (Luo and Stopa,2004). 1992). TRH has been shown to have neuroprotective functions in primary neuronal cultures (Koenig et al, CHOLECYSTOKININ 1996) due, at least in part, to its potent inhibition of GSK-3 via PKC-mediated phosphorylation (Luo and Cholecystokinin (CCK), a peptide originally Stopa, 2004). The neuroprotective activify of TRH, . discovered as a -like molecule of the together with the known facilitation of cholinereic gastrointestinal tract, plays an important role in

i I t. I l' $ ta f'": t I f I 40 C. SEVERINI ETAL.

'a the release of, pancreatic enzymes, gall bladder It was demonstrated that CCK enhances memory contraction, and gastric motility. As recently retention and protects cholinergic neurons against reviewed (Beinfeld handbook 2013 ,738-743), CCK basal forebrain lesions (Sugaya et al, 1992). is one of the most abundant and widely expressed Alterations in the CCK system have also been neuropeptides in the brain, essentially as CCK8 amide, correlated with AD. Indeed, despite the content of in the sulphated form. CCK shares the same five CCK in the brain of AD patients being generally carboxyl-terminal amino acids with gastrin, which is relatively unchanged, in the most severe cases it was believed to have evolved from CCK. CCK has been found to be reduced (Perry et al, 1981; Sagar et al, shown to be present in microgram quantities in the 1984; Mazurek and Beal, l99I; Lófberg et al, 1996). brain, distributed in high levels in the hippocampus, In addition to reduction in CCK content in brain amygdala, septum, olfactory tubercles, caudate tissues, neuritic plaques in the AD brain have been nucleus and the hypothalamus, ventral tegmental demonstrated to contain CCK (Struble et al, 1987; area and substantia nigra (Schiffrnann et al, 1991) Perry et al, 1981). (Fie. 1). Moreover, the CCK receptors are also affected, CCK co-localizes with several key as a significant down-regulation was demonstrated neurotransmitters and neuromodulators. of both CCKA and CCKB receptors in the brain of prevalently y-aminobutyric acid (GABA), as well patients withAD and with mild cognitive impairment, as endocannabinoids, dopamine, and suggesting that these receptors could play a role inAD vasoactive intestinal peptide (Hokfelt et al, 1980; development (Hokama et al,20l4; Lin et al,20I4). Somogyi et al, 1984; Kosaka et al, 1985; Katona et Alterations in CCK content have been found also al,1999), providing additional evidence for its wide- in AD animal models. Unlike the decrease in peptide ranging role in physiological functions in neuronal levels reported in studies onAD humanbrain, increased networks. levels of CCK were demonstrated in hippocampus CCK interacts with two G-protein (Go and G,,) and cortex, as well as in neuritic plaques of 18- and receptors, activating a variety of intracellular signal 26-month-old transgenic mice overexpressing V 7 17 F transduction pathways (Williams et al, 2002). Protein human beta-amyloid precursor protein (Diez et al, kinase C activation is the main signaling for CCKA 2000). By contrast, inAPP23 mice a decrease in CCK receptor (CCKA-R), while adenylyl cyclase is for immunoreactivity was shown in hippocampal mossy CCKB receptor (CCKB-R). CCKA-R is relatively fibers (Diez et al, 2003). specific for sulphated CCK8, while CCKB-R (identical to the gastrin receptor) interacts also BRADYKININ with un-sulphated CCK8, CCK4 and gastrin and represents the main CCK receptor in the brain. CCK The bradykinin (BK) and kallidin, also acts as an excitatory neurotransmitter modulating called Lys'0-BK, ate oligopeptides released in the release and function of other neurotransmitters the plasma or interstitial fluid after the cleavage and is involved in diverse normal behaviors, such of by , a family of serine as learning and memory feeding, nociception and proteases. Plasma and tissue kallikrein satiety. The central importance of CCK in neuronal I (KKl) are the main enzymes involved in kinin nefworks is atrso reflected by its involvement in a source in blood and tissue, respectively. -1 variety of neurological and neuropsychiatric disorders gene (KNG 1) is a glycoprotein that contains the BK including anxiety, panic attacks, schizophrenia and sequence in its mid portion (Fig. 1). epilepsy (Lee and Soltesz, 20II). Several reports Kinins are potent vasodilators, promote natriuresis suggest a modulatory role of CCK in memory and diuresis, and have beneficial cardiovascular processing (Flood et al, 1987, Kovàcs and De Wied, effects, however, they also promote pain and 1994), an aspect of crucial relevance in AD, in which inflammation (Bhoola et 41, L992). Components memory and other cognitive functions are impaired. of the kallikrein-kinin system are present in blood, European Journal of Neurodegenerative Diseases 4T heart, aoÍta, brown adipose tissue, adrenal and Additionally, an activation of the contact/kinin lung. In the brain, they are localized in the cerebral system in CSF of patients withAD has been reported cortex, brain stem, cerebellum, hypothalamus, as the result of an anionic interaction of residues hippocampus, and pineal gland, among others. They ' pithin the region 1-11 of AB,_0, with factor XII, are found surrounding blood vessels, in neurons and inducing kallikrein generation. This finding seems glial cells (Raidoo and Bhoola, 1998). to be characteristic for brain ofAD patients, since in In human plasma, BK is rapidly metabolized the CSF of patients with neuroimmune inflammatory by kininases, among which aminopeptidase p disease (multiple sclerosis, chronic inflammatory and carboxypeptidase N. However, the major demyelinating polyneuropathy) there was no degradation pathway of BK involves also kininase II evidence of increased cleavage of high molecular and angiotensin-converting enzyme (ACE), which weight kininogen (Bergamaschini et al, 2001). is also responsible for the conversion of inactive According to Farrall et al, (2009), the frontal angiotensin I into the vasopressor angiotensin II cortex of patients with AD shows high levels of (Boola et al, 1992). plasma kallikrein as well as its mRNA. This finding Biological effects of kinins are produced by and the high enz.yme activity suggest that kinin activation of two transmembrane receptors coupled production could influence cerebral blood flow and to G proteins (Go and Gq), namely B I and B2 vascular permeability altered in AD. receptor (Regoli and Barabe, 1980). Most actions A more direct evidence for BK involvement in of kinins are mediated by 82 receptor, which has AD pathology was demonstrated analyzing BK high affinity for BK and is considered a constitutive release and its processing in brain and cerebrospinal receptor (Regoli et al, 1998). On the other hand, Bl fluid (CSF) of rats infused chronically with Ap receptor possesses higher affinity to des-Arge-BK (Iores-Margal et al, 2006). In CSF of animals and Lys-des-Arge-BK and has limited distribution in infused with Ap, BK concentration was significantly tissues under physiological conditions. However, it increased, however, in the brain of Ap group, only is mainly expressed in pathological conditions such a BK fragment was detected. These results suggest as chronic inflammation, infection or injury (Regoli that the kallikrein-kinin system is activated in and Barabe, 1980). Activation of kinin receptors this AD animal model, and that BK is efficiently induces phospholipase C stimulation and promotes inactivated by kininases in brain. Since it was intracellular calcium mobilization, as well as release reported that in cell cultures BK can increase alpha- of nitric oxide (NO), especially on neurons and blood secretase processing of APP, inducing decreased vessels (Marceau, Regoli, 2004). AF,_oo, a major constituent of amyloid plaques, The role exerted by kallikrein-kinin system in BK inactivation could ultimately contribute to the AD and other neurological disorders was recently increased senile plaque deposits in the rat brain reviewed (Viel and Buck ,2011; Naffah -Mazzacoratti (Nitsch et al, 1998). et al, 2014). It was demonstrated that proteolytic In addition to the variations in kallikrein-kinin enzymes levels are altered. in AD (Ladror et al, enzymatic system, also BK receptors expression 1994). Among these, intracerebral kallikrein seems appeared to be modified in AD, mainly related to play an important role in the pathogenesis of AD, to neuroinflammation (Marceau and Bachvarov, as demonstrated by a reduced kallikrein-like enzyme 1998, Viel and Buck, 2011). Indeed, in cultured activity, due to a reduction in the gene expression in skin fibroblasts from AD patients an increase in the cerebral tissue of AD patients (Aoyagi et al, 1990). number of BK receptors was reported (Huang et al, r,i Likewise, increased expression of kallikrein 10 1995), as well as biochemical abnormalities in B2 il ::i and kallikrein 6 were observed in CSF, plasma and receptor functions (Jong et al, 2002). 'l,.i whole ,,.1 blood of patients with AD, showing a strong Moreover, increased expression of B I receptor i]i relationship between the kallikrein-kinin system and was found in hippocampal astrocytes of AD mice. brain degeneration (Diamandis et a1,2000; 2004). In the same work, the ability of BIR antagonists 42 C. SEVERINI ETAL.

to abrogate amyloidosis and cerebrovasculàr and activity ofACE inhibitors inAD (Zouand Michikawa, memory deficits was demonstrated, providing 2008). ACE inhibitors (such as enalapril, ramipril evidence for a harmful role for B 1R in AD and many others) are well-established as important pathogenesis (Lacoste et al, 20L3), despite other antihypertensive drugs, liable to block conversion experimental data indicating that in Tg-SwDI mice of inactive angiotensin I into the vasopressor BlR activation plays an important role in limiting angiotensin II. However, they also increase half- the accumulation of AB in AD-like brain (Passos et life of BK, enhancing levels of circulating BK and al, 2013). potentiating BK receptors. At least in vivo, ACE It was also shown that chronic i.c.v. injection ofAB,_ inhibitor activity is mainly mediated byB2receptors, oopromotes significant increase in densities of kinin B 1 as demonstrated by the inhibitory effect exerted by and 82 receptors, mainly in brain regions related to 82 receptor antagonists (Marceau and Regoli,2004). cognitive behavior (Viel et al, 2003). Nevertheless, a The ability of ACE inhibitors to modulate the kinin single i.c.v. injection of aggregated AB,_oo induced an system could be responsible, at least in part, for the increase in B I receptor expression in hippocampus, observed neuroprotective activity. but did not modify B2 expression in the same area (Prediger et al, 2008). These variable results suggest CHROMOGRANIN/SECRETOGRANIN FAMILY that the involvement ofthe kinin system inAB toxicify could be a function of the quantity ofAB, as well as a The chromogranin/secretogranin family function of the exposure time of tissue to the peptide. represents an extended but functionally conserved On the whole, while BlR is certainly involved in family of ,oroteins, including chromogranins neuroinflammation related to AD, B2R preferentially (chromogranin A and chromogranin B), seems to mediate neuroprotective effects. Activation of secretogranins (secretogranin II and secretogranin B2R by BK was demonstrated to reduce inflammation III), and related proteins (782, NESP55, proSAAS, and neuronal death (Noda et al, 2007) and to promote and VGF). They are localized in secretory vesicles neurogenesis (Trujillo et al, 2012). Furthermore, it and are variously distributed in endocrine, neuronal was shown that activation of 82 receptors, but not Bl and neuroendrocrine cells, as well as in the immune receptors, up-regulates mRNA for nerye growth factor system and occasionally in other tissues, subserving (NGF) in glial cells, establishing a neuroprotective essential roles in the regulated secretory pathway condition (Noda et al, 2007a). that is responsible for controlled delivery ofpeptides, New advance in the role exerted by BK hormones, neurotransmitters, and growth factors. In receptors in AD animal models is represented the brain, they are widely localized in different areas by the availability of B 1 and 82 knockout mice. (Fig. 2). Following Ap infusion, B 1 knockout mice did not The first granins identified were chromogranin show any difference in memory behavior compared A (CgA) and chromogranin B (CgB), purified to control animals with the same treatment, while 82 from adrenal medulla, but other proteins were knockout mice resulted in a significant reduction in successively added to this family, all sharing some memory consolidation (Amaral et aI, 2010). These commune features. They are large acidic proteins, data demonstrate that, following chronic infusion sometimes glycosylated or sulphated, having the with AB, 81 receptor could play an important role ability to bind calcium and, although very soluble, in the neurodegenerative process, while 82 receptor to aggregate in the acidic compartment, inducing the could have a neuroprotective role. This is further formation of dense core granules in the presynaptic confirmed by the increased number of AB plaques structures. Granins regulate different functions, found in 82 knockout mice infused with AB, pointing acting as chaperons for protein sorting, modulating toB2 receptor as a potential therapeutic target in AD prohormone convertase activity and regulating (Caetano et al, 2015). secretory vesicle content release. Moreover, through Another point to be considered is the potential the secretory pathway, most ofthem are proteolitically European Journal of Neurodegenerative Diseases 43

cgA 439 aa KW CgB GTT aa @ Sgll 617 aa ffi Sgttt 468 aa ffi 782 272aaffi @ffi VGF 615 aa @ PToSAAS z6aaa ffi

Fig. 2. Granin precursors sequence and brain distribution. The scheme represents the approximate length and the number of amino acids of the preaffsor of CgA, CgB, SgII, SglII, 782, VGF and PToSAAS. The leader signalfor secretory protein is indicated as light shadowed block. The black blocks indicate the biological active fragments identified. In the right panels are shown images of in situ hybridizationfor the corresponding gene expression in mouse CNS (Sourcefrom Allen Brain Atlas, mouse. brain-map. org).

processed in different biological active peptides the processing of these large precursor molecules that are stored in large dense core granules and in many different neuropeptides, it is difficult to released upon secretory stimulation (for extensive recognize the precise identity of these fragments review see Bartolomucci et al, 2011). Despite because often Authors refer to the whole precursor. repeated attempts to demonstrate the existence of cognate receptors, no evidence for specific granin a) Chromogranin A receptors, or the precise mechanisms of action has CgA, a 439-amino-acid protein, was the first been produced to date. Since granins are largely identified granin and the most extensively studied distributed in the CNS and are involved in synaptic (Winkler and Fischer-Colbrie, 1992). CgA was functions, several studies investigated the potential postulated to be an immunostimulator in AD, utility of granins as diagnostic biomarkers for AD contributing to neuroinflammation started by AF and other neurodegenerative diseases (Bartolomucci peptides (Heneka et à1, 2010). Indeed, intense et al. 20ll Willis et al. 2011). However. due to staining for CgA was demonstrated in about 30% 44 C. SEVERINI ETAL.

of Ap plaques in AD cortical samples, frequently conserved across evolution and, together with CgB surrounded by hyperactivated microglia (Marksteiner is the major soluble constituent of the large dense etal, 2002; Rangon etal, 2003;Lechner etal, 2004: core vesicles of presynaptic structures (Fischer- willis et aI, 20lr). cgA increase in neuritic plaques colbrie et al, 1995). Several studies showed an correlates with an increase in catestatin (cS! an increased SgII immunostaining in amyloid plaques, internal fragment of CgA), able to activate pro_ while a significant reduction in immunoreactivity caspase-l (Wu etal, 2013). was described in different brain areas ofAD patients There is evidence that cgA activates microglia (Kaufmann, 1998; Marksteiner et al, 2002;Lechner to a reactive phenotype and stimulates the release of et al, 2004). Alterations'in SgII content have been microglial cytotoxins, suggesting that this peptide may reported also in animal models of AD. Indeed, in contribute to the continued and neurotoxic activation brain of transgenic mice overexpressing human of microglia in AD (Lechner et 2004). al, APP75 1 with the London (V7 t7I) and Swedish CgA staining increase in the amyloid plaques 6670Mn{671L) mutations, about 40%of amyloid_ inversely correlates with cgA levels found in the csF, beta plaques were associated with SgII, however no significantly reduced in patients withAD or taupathies immunostaining reduction was observed in specific (Blennow et al, 1995; Eder et al, l99g; Simonsen et brain areas, compared to controls, as otherwise aI, 2007; Perrin et al, 20lt; Jahn et al, Z0ll; Mattson reported in AD patients (Willis et al, 2009). et al, 2012; Paterson et al, 2014; Wildsmith et al, From SgII processing three major products are 2014). Among CgA derived peptides, only Serp in (26 obtained: Secretoneurin (SN , 33 aa sequence, near aa at c-terminal) showed a neuroprotective activity, the N-terminal portion), EM-66 (central portion) as demonstrated in vitro, in AtT20 neuronal cell line and Manserin (40 aa sequence, near the c-terminal (Koshimint et ar, 20ll). To date, no neuroprotective regiop). consistent with the decreased rever of cgA functions have been identified vasostatin, for and CgB found in in CSF of AD patients, also SN Pancrestatin and chromacin, the other major forms or other SgII fragments were found to be reduced of the CgA-derived neuropeptides, endowed with (Eder et al, 1998; Matsson et aI, 2010; Spellman neuroendocrine activities. et al, 2015). In addition, SN neuropeptide was demonstrated to promote in vitro and in vivo b) Chromogranin B neuroprotection after oxygen/glucose deprivation, CgB, a pre-pro-protein of 677 amino acids, shares suggesting an anti-apoptotic activity through Jak2l several features with cgA, including wide expression Stat3 signaling pathway (Shyu et al, 200g). throughout the endocrine and nervous systems, acidic protein backbone, random-coil structure, d) Secretogranin III and heat stability. CgB is abundantly expressed SgIII is an acidic 468 amino acid secretory in many neurons and peptidergic endocrine cells protein, well conserved during evolution, from (Bartolomucci et al, 2011). mammals to fish. It has been identified as a specific Immunostaining of post-mortem brains from AD binding protein for CgA, suggesting that it can play a patients showed positive CgB reactivity in amyloid central role in secretory granule biogenesis (Hosaka plaques, more prominent in hippocampal regions and Watanabe, 2010). (Marksteiner et al, 2002; Lechner et al, 2004;Willis Although different fragments of SgIII have et al, 2008, 20ll), while levels of CgB peptides in been detected in several neuroendocrine cell types CSF of AD patients were significantly decreased, from various species (Holtious et al, 1996)t, no compared to control subjects (Eder et 199g; al, biologically active peptides derived from sgIII have Mattson et al, 2010;2012;2013). been described. In the cerebral cortex of AD patients increased levels of sgIII were observed in dystrophic c) Secretogranin II neurites surrounding amyloid plaques (plà et al, SgII is a 617 amino acid pre-pro-protein highly 2013). Additionally, SgIII was detected in CSF bur European Journal of Neurodegenerative Diseases 45

no significant differences between AD patients and that N-proSAAS or proSAAS-like molecules are control subjects were reported (perrin et al, 201l). trapped within the tau fibrils and accumulated in tau inclusions in the AD patients biains (wada et e) 782 b " 81, 2004). In addition, in the brain of l2-month_old The smaller granin 7F.2 (212 amino acids) is APdE9 mice, and in the cortex of human AD affected perhaps the most evolutionarily conseryed member brain, proSAAS immunoreactivity co-localizes with of the granin family, containing a c-terminal peptide amyloid plaques deposits (Hoshino et al, 2014\. acting as inhibitor of the prohonnone convertase 2 Like other granins, proSAAS fragments were (PCz), responsible for the proteolytic processing of found decreased in csF ofAD patients in respect to many precursor proteins, among which neuropeptides healthy subjects, suggesting that they could represent, precursors (Mbikay et al, 2001). Alterations, often together with other neuropeptides, biomarkers for conflicting, in PC2 and 7F'2 Ievels have been shown AD (Jahn et al, Z0ll). As previously reported for in AD brain, related to a dysreguration in the rever 782, additional evidence for the role of proSAAS in of different neuropeptides. A marked decrease in AD was demonstrated by the amyloid anti-aggregant the ratio of the PC2 precursor to the total enzymatic activity of an internal prosAAS fragment (prosAAS pool was observed in the frontal cortex of AD 97-180), shown to efficiently prevent the fibrillation patients, corresponding to an increase in the binding ofAp,-or, in vitro (Hoshino et al, 2014).In the same protein 7B.2 (Winsky-Sommerer et al, 2003). On study, the Authors reported that the recombinant, the contrary other studies reported increased levels as well as the endogenously synthesized proSAAS, of PC2 in AD brain, while no differences were was able to prevent the neurotoxic effect ofAB,_., in detected in the levels of 782 (yakovlera et al, 2007). Neuro2a cells. Likewise, no significant differences were found in the 782 immunoreactivity in various brain regions g) VGF obtained from patients with AD and from control The large secretogranin VII, early named VGF, subjects (Iguchi et al, 1987). ii is a 615 amino acid protein precursor of several More recently, in the hippocampus and substantia biological active peptides. Adozen of them have been nigra of human AD-affected brains, as well as in detected in cSF of patients with neurodegenerative the brains of APP/PSENI mice, 7Fi2 was found diseases. In cSF ofAD patients, both an N-terminal highly co-localized with AB plaques and a-synuclein fragment and an intemal peptide (VGF 365-375) deposits (Helwig et al, 2013).In the same work it were found to be decreased (carrette el al, 2003; was demonstrated that 7F.2 efficiently prevents in Jahn et al, 20ll; Wujte et al, 2012; Spellman et al, vitro fibrillation and formation of Ap aggregates, 2015; Hottla et al, 2015; Hendricson et al, 20IS), establishing this neural protein as an anti-aggregation suggesting that vGF could be a potential biomarker chaperon associated with neurodegenerative diseases. for this disorder. Moreover, in parietal cortex from In addition, recombinant 782 efficiently blocked the patients with AD, a reduction in different VGF neurocytotoxic effect ofAp,_n, significantly increasing peptides was shown (Cocco et al, 2010), whereas cell viability of Neuro-2A cells (Helwig et aI, 2013). an increase of vGF expression was reported in peripheral T cells in patients with AD, compared to PToSAAS fl aged healthy controls (Busse et al, 2015). PToSAAS granin is a 260 amino acid precursor Interestingly, it was demonstrated that VGF_ protein suggested to function as peptide precursor derived peptides exert important neuronal only in higher vertebrates (Bartolomucci et à1, stimulatory activity. TLep_62 (C_terminal VGF 20ll). Like 782, it has been suggested to regulate the peptide) has an antidepressant activify (Lin et al, activify ofthe pro-hormone convertase pc I (Lee et al, 2014), increases neuronal electrical excitabilify in 2004). The potential role exerted by proSAAS in the hippocampus neurons (Takker-Varia et al, 2007), pathogenesis of AD was suggested by the evidence while TLQP-21 (a smailer c-terminal vGF peptide) 46 C. SEVERINI ETAL.

protects from apoptosis cerebella, grun.rl. cells in patients with dementia of the Alzheimer type. Biot after potassium deprivation (Severini et al, 2003). Psychíatry 33(4):267 -7 t. Moreover, in primary cortical and hippocampal Amaral FA, Lemos MT, Dong KE, Bittencourt MF, cell culfures, TLQP-21 showed a neuroprotective Caetano AL, Pesquero JB, Viel TA, Buck HS(2010) activify against AB toxiciry while other fragments Participation of kinin receptors on memory impairment had minor or no neuroprotective effect (possenti R., after chronic infusion ofhuman amyloid-b etal-4}peptide unpublished data). in mice. lt{europeptides 44(2):93-1-. So far, only for TLQP -21 peptide a receptor has been recently discovered, identifuing the variously Anand R, Gill KD, Mahdi AA (2014) Therapeutics Alzheimer's past, distributed complement C3a receptor- l (C3AR1) of disease: present and future. Neuropharmacology as a target for TLQP-2l (Hannedouche et al, 2013: 76 pt A:27-50. Cero et al, 2014). Aoyagi t Wada ! Nagai M, Kojima F, Harada S, Takeuchi ! Takahashi H, Hirokawa K, Tsumita T (1990) CONCLUSIONS Deficiency of kallikrein-like enzyme activities in cerebral tissue of patients with Alzheimer's disease . Experientia AD has become a great i clinical problem in our society a6Q):94-7. and the prevalence of AD is likely to increase among Bartolomucci A, Possenti R, Mahata SK, Fischer_ the aging population worldwide. To date, no treatment Colbrie R, Loh YP, { Salton SR (2011) The extended granin had been found that could slow down the progression family: structure, function, and biomedical implications. of the disease or that could prevent cholinergic cell Endocr Rev 32(6): 7 55-97 . death, as the current therapeutic approach to AD is of Bennett GW, Ballard TM, Watson a symptomatic type. Since the pathology ofAD is very CD, Fone KC (1997) Effect of neuropeptides on complex and different pathomechanisms are involved, cognitive function. Exp Gerontol 32(a-l: 451 the ultimate goal of a sustainable disease-modifying -69. treatment in AD is to slow down disease progression Beinfeld MC (2013) CCK. Handbook of Biologicaily by addressing the neurodegenerative processes, Active Peptides, second edition, edited by Abba J. KasîÌn. acting at multiple pathways. The role exerted by chopter 99: 738-743. neuropeptides seems particularly interesting since Bergamaschini L, Donarini C, Gobbo G, parnetti L, they are generally neuroprotective, widely distributed Gallai v (2001) Activation of complement and conract in brain areas responsible for learning and memory system in Alzheimer's disease. Mech Ageing Dev IZZ: processes, and their levels are altered in both human l 97 r -1 983. disease and in animal experimental models. Since Bhoola KD, Figueroa CD and Worthy K (lgg2) neuropeptides could represent biomarkers of disease Bioregulation of kinins: kallikreins, kininogens progression, this seems of great potential utility for and kininases. Pharmacol Rev 44: l-80. AD because of inherent difficulties assessing brain function and finding a diagnosis in this pathology. In Biggins JA, Perry EK, McDermott JR, Smith AI, addition, convincing evidence has been provided for Perry RH, Edwardson JA (1983) post morrem levels of the protective role of several neuropeptides against thyrotropin-releasing hormone and neurotensin in the neurode generati amygdala in Alzheimer's on b oth i n v i îro and i n v iv o,identi ff ing disease, schizophrenia and neuropeptide receptors as potential therapeutic targets. depression. J l'{eurol,Sci 58: ll7-22. Blennow K, Davidsson p, Wallin A, Ekman R (1995) REFERENCES chromogranin A in cerebrospinal fluid: a biochemical marker for synaptic degeneration in Alzheimer's disease? Albert M, Jenike M, Nixon R, Nobel K. (1993) Dementia 6(6):306- I l. Thyrotropin response to thyrotropin-releasing hormone Bouras c,de st Hilaire-Kafi S, constantinidis J (19g6) European Journal of Neurodegenerative Diseases 47

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