CNS & Neurological Disorders - Drug Targets (Formerly Current Drug Targets - CNS & Neurological Disorders) - Volume 10, Issue 3, 2011

Putting a Finger on Neurotrophic Protein Therapy in Parkinson's Disease Parkinson's disease (PD) was first described in the essay entitled “An Essay of the Shaking Palsy” by James Parkinson in 1817. PD is the most common neurodegenerative movement disorder, whose neuropathological hallmarks are characterized by progressive and profound loss of neuromelanin-containing dopaminergic neurons in the substantia nigra pars compacta with the presence of eosinophilic intracytoplasmic, proteinaceous inclusions termed Lewy bodies and dystrophic Lewy neurites in surviving neurons. Although neuronal cell loss in the substantia nigra pars compacta is pronounced, there is widespread neurodegeneration in the CNS with the pars compacta being involved in midstages of the disease. Clinical manifestations of this complex disease include motor impairments involving resting tremor, bradykinesia, postural instability, gait difficulty and rigidity, along with non-motoric symptoms like autonomic, cognitive, and psychiatric problems. As with many multifactorial diseases, the incidence of PD increases with age. Because of an aging population, improved diagnosis, and prolonged survival, especially in developing countries, the number of patients suffering from PD is predicted to double by the year 2030. The power of neurotrophic factors to regulate neuronal cell survival in the developing nervous system and to promote also survival after injury or protect neurons in toxin-mediated disease models in animals has encouraged the idea that such proteins could be harnessed for the treatment of neurodegenerative disease. In the case of dopaminergic neurons, glial cell line-derived neurotrophic factor (GDNF), a member of the basic fibroblast growth factor superfamily, given intracerebrally has neuroprotective and neurorestorative effects in neurotoxin-induced rodent and non-human primate models of PD. In spite of extensive positive preclinical data and encouraging results from phase I clinical trials, results from phase II studies have been disappointing. Indeed, neurotrophic factor treatment of CNS diseases presents an especially complex problem, since these polypeptides have poor pharmacokinetics and bioavailability and are not able to cross the blood-brain barrier. At the same time, excessive levels of a neurotrophic protein may provoke deleterious side-effects, as evidenced by toxicity (including multifocal cerebellar Purkinje cell loss) observed in rhesus monkeys that received intraputamenal infusion of high concentrations of GDNF. A novel approach to this problem has recently been reported by Laganiere and colleagues, who explored the possibility of engineering zinc finger protein transcription factors (ZFP TFs) that activate the expression of the endogenous GDNF gene. Transcription factors based on Cys2-His2 ZFPs can be engineered to specifically target virtually any gene. By targeting the endogenous gene and exploiting the native promoter, which imposes a physiological upper limit on the level of gene expression from each allele, a ZPF can produce sufficient, but not supraphysiological levels of the therapeutic protein needed to achieve both long-term efficacy and safety. Laganiere et al. designed a human GDNF ZFP TF (hGDNF-ZFP) that recognized a site that is fully conserved between human and rhesus macaque. Having confirmed that hGDNF-ZFP activated rhesus GDNF in rhesus macaque cell lines, the authors then designed and assembled a panel of ZFP activators specific to the rat GDNF promoter. Using an adeno-associated viral vector serotype 2 as the delivery vehicle, the engineered transcription factor that gave the highest levels of rat GDNF activation was tested in the rat 6-hydroxydopamine model of PD. Vector was infused bilaterally into the striatum by convection-enhanced delivery. The modest (∼60%) overall increase in striatal GDNF levels provoked by the ZFP was sufficient to provide marked improvement in all behavioral tests performed. Moreover, functional neuroprotection by the GDNF activator was supported by immunohistochemical demonstration of preservation of tyrosine hydroxylase-positive nigrostriatal neurons. While activation of endogenous GDNF was adequate to protect again neurotoxin lesion in rat, demonstration of efficacy and safety in a nonhuman primate model of PD will be a prerequisite before advancing the ZFP-based approach to the clinic. The dual species-specific GDNF activator should facilitate such studies. In addition, convection-enhanced delivery is expected to provide a more complete coverage of the putamen, unlike previously used infusion protocols. Engineered ZFP activators that drive specific activation of endogenous GDNF combined with highly efficient convection-enhanced delivery may well offer hope to realize the therapeutic potential of GDNF.

Soon after its discovery during early seventies, prolyl oligopeptidase (abbreviated PREP, POP, PO, or PEP) was shown to cleave neuroactive peptides. This finding hinted that the peptidase would have an important role in controlling the physiological neuropeptide levels and thus, its inhibition would have a direct consequence on central nervous system function modifying mood, behaviour, perception and its processing, or memory. This rationale guided the first efforts to develop specific inhibitors already during the late seventies. Subsequent to this, and after it was described that PREP inhibitors would counteract memory loss caused by chemical insults or brain ischemia, the search for potent compounds boomed, particularly in Japan. Indications that PREP inhibitors were neuroprotective, further fueled the interest of pharmaceutical companies to dedicate resources towards the development of a drug efficient for brain PREP inhibition. Patent applications were then filled to cover compounds indicated for dementia and neurodegeneration. However, the role of PREP in neuropeptide metabolism has been difficult to establish, and the search for the physiological relevance of the peptidase has been a task difficult to achieve. This special issue of CNS & Neurological Disorders-Drug Targets aims to provide a critical review of the research on PREP and delineate new ways this intriguing enzyme might be working not only in the brain, but also in the periphery. Contributions to this issue revise basic information on PREP structure (see Rea and Fulop) and its relation with function (see van Elzen and Lambeir). Essential to understand PREP's function is the determination of its physiological substrates (see Tenorio-Laranga, et al.) on one hand, as well as the physical and functional localization of PREP in the brain (see Peltonen, et al.), on the other. More evidence is emerging pointing to PREP having roles connected to intracellular signalling and transport (see Harwood, and also Moravski, et al.) in which direct protein interactions ( see Lambeir) might be of relevance. The relationship of PREP and disease is also reviewed, not only in connection with dementias, but also with pathologies with an inflammatory component (see Penttinen et al). To the eye of a molecular scientist, the size and structure of PREP indicates a function beyond a simple peptidase. Serine proteases have a small, compact α/β-hydroxylase folded structure, but PREP is provided with an extra propeller domain of uncertain function. It seems of some consensus that PREP possesses a non-hydrolase function. This is also strongly suggested from the study of a structurally related protein, PREPL, of an apparently non-peptidase activity but with profound health consequences over its genetic disturbance (see Boonen, et al.). With regard to the drug target importance of PREP, it seems even more interesting scenarios are emerging on this respect and more research might be stimulated from now. In this enterprise, funding, as the one obtained from the 7th Framework programme of the European Commission for most of the research on PREP described in this issue, has been fundamental, as well as the interest of CNS & Neurological Disorders-Drug Targets editorial managing to disseminate the outcome of this research.

Prolyl oligopeptidase (POP) belongs to a unique class of serine proteases. Based on extensive enzyme kinetic measurements it has become clear that POP acts in a multifaceted way. This is reflected in the complex behavior in different reaction conditions with different substrates. Also the typical structural architecture of POP, with the active site located at the interface of the catalytic domain and the β-propeller domain, has instigated many researchers to speculate about the mechanism of functioning. The latest developments support the idea of extended conformational changes upon substrate binding. In this review the plethora of available information is assembled into a coherent and stepwise description of the structural composition of POP. In one aspect the composition and contribution of structural boundaries at the active site are described. Attention is focused on the catalytic components and the features that are presumed to confine the substrate specificity. Complementary to this, the specificity of POP towards the residues surrounding the scissile bond is described by means of a consecutive evaluation of the preferred physico-chemical properties. Together, these two approaches may facilitate a better understanding of the concepts that determine catalytic behavior of POP in physiological conditions.

Prolyl oligopeptidase or prolyl endopeptidase (PREP; EC 3.4.21.26) is an atypical serine protease that hydrolyses peptides and peptide hormones after proline in peptides up to around 30 residues long. Evidence suggests an involvement in learning and memory, and the enzyme is implicated in diseases including amnesia and depression. The first crystal structures determined, of the porcine enzyme, provided direct insight into the mechanisms of substrate size selectivity, substrate specificity, and catalysis. However in these structural studies the enzyme is in a closed state, even in the absence of ligand, leaving questions as to how substrates and products can enter and exit the enclosed central cavity that houses the active site. More recent crystal structures of bacterial PREP have captured the enzyme in an open state, revealing the true extent and nature of the structural dynamics involved, and illuminating an induced fit mode of catalysis and regulation. Molecular modeling has further contributed to our understanding of the conformational changes that occur during catalysis. Here we review the data that has led to our current understanding of the structure and dynamics of this biologically and pharmaceutically important enzyme.

Prolyl oligopeptidase (POP), is an 80-kDa serine protease that hydrolyzes peptides smaller than 30-mer at the carboxyl side of an internal proline-residue. POP is commonly believed to cleave a number of neuropeptides claimed to be involved in learning, memory and mood. While the support to the neuropeptide cleavage theory has been declining, new data suggest novel functions for POP, e.g. as a regulator of protein secretion, α-synuclein aggregation and cell proliferation/differentiation. Intriguingly, many of these novel functions may not depend on the hydrolytic activity of POP. One of the new roles is an involvement of POP in neurotransmission. Indeed, POP has been associated with a variety of different neurotransmitters. According to our previous results, POP is located in GABAergic and cholinergic neurotransmitter systems and in short glutamatergic projection neurons between cortex and thalamus. Based on these findings, POP may be involved in inhibitory and excitatory signal transmission and in the thalamocortical and corticothalamic signalling in the brain. It has also been shown that POP inhibition can affect the functions of neurotransmitters and that the ligands of neurotransmitter receptors can alter the activity of POP. Thus, there is a connection between the neurotransmitters and POP, although the mechanisms and the functional significance of this linkage are still unknown. Here, we will review the data about the connections of POP with conventional neurotransmitter systems and provide an overview of the role of POP in neurotransmission.

Prolyl oligopeptidase (POP) is a serine protease that cleaves peptides shorter than 30-mer at the carboxyl side of an internal proline. POP has been proposed to be involved in some pathologies including mood disorders and neurodegenerative diseases. However, the physiological role of POP remains unknown. To validate POP as a drug target, it is essential to obtain a thorough understanding of its function in vivo. Identification of physiological substrates and products of POP is an important step towards this goal. Recent peptidomic studies have revealed some biological substrates of POP and have given information about the in vivo consequences of POP inhibition. The aim of this review it is to evaluate new advances in this research area and to critically confront these data with initial conclusions and proposals. It seems that substantial activity of POP occurs intracellularly in contrast to the previously proposed role of this peptidase on the direct degradation of extracellular neuropeptides.

Prolyl endopeptidase (E.C. 3.4.21.26, PREP) also known as prolyl oligopeptidase is an enzyme which cleaves several peptides at the carboxyl side of proline residues. Since brain contains relatively large amounts of this enzyme and because of its substrate specificity it has been suggested to play a role in the metabolism of neuropeptides, acting both on their maturation and their degradation. The final step of neuropeptide processing occurs in the synaptic vesicles and degradation of most of these peptides takes place in the synaptic cleft. Thus, a localization of PREP in these cellular compartments appears to be feasible. Here we summarize recent data and provide novel evidence for the subcellular localization of PREP. Most importantly, immunocytochemical double labelling, confocal laser scanning and electron microscopic procedures as well as functional assays strongly suggest a role for PREP in intracellular transport mechanisms and in protein secretion.

Inhibition of prolyl oligopeptidase (PO) elevates inositol phosphate (IP) signalling and reduces cell sensitivity to lithium (Li+). This review discusses recent evidence that shows PO acts via the multiple inositol polyphosphate phosphatase (MIPP) to regulate gene expression. As a consequence, PO inhibition causes both a transient, rapid increase in I(1,4,5)P3 and a long-term elevation of IP signalling. This pathway is evolutionary conserved, being present in both the social amoeba Dictyostelium and human cell systems, and has potential implications for mental health.

Inhibitors of prolyl oligopeptidase have been reported to be neuroprotective, especially in memory loss caused by lesion or disease. This enzyme has also been implicated in neurodegeneration. Although it was initially thought that prolyl oligopeptidase functioned to directly control of neuropeptide levels, emerging evidence points out in part that this peptidase modulates peptides which in turn regulate inflammatory responses. Here we review the recent literature which indicates a direct involvement of prolyl oligopeptidase in several inflammatory diseases. Neuroinflammation generates neurotoxins with a relevant role in neurodegenerative diseases, and it is within this toxin generation where prolyl oligopeptidase might have a role.

Prolyl oligopeptidase (PO) interacts with α-synuclein in vitro. It is a weak interaction that induces a nucleation prone conformation of α-synuclein. PO accelerates aggregation and fibril formation of α-synuclein in a process that can be reversed by specific inhibitors and is also influenced by an impairing mutation in the PO active site. There is evidence that PO and α-synuclein also interact intracellularly, especially in conditions where the expression of α-synuclein is high. Specific PO inhibitors reduce the number of cells with α-synuclein inclusions in a cellular model of Parkinson's disease. If these interactions also exist in the human brain, PO may be a target for the treatment of Parkinson's disease and other synucleinopathies. Whether PO also contributes to the normal physiological functions of α-synuclein remains an open question, but there are some intriguing parallels between the proposed functions of both proteins that deserve further investigation.

Deletion of the Prolyl Endopeptidase-like (PREPL) gene has been described in three contiguous gene deletion syndromes at the 2p21 locus and current developments in high resolution microarrays and whole genome sequencing will no doubt soon result in the identification of isolated PREPL deficiency. But by comparing the differences in phenotypes with the number of genes deleted, the contribution of PREPL deficiency can already be deduced. Homozygous or compound heterozygous loss of PREPL is predicted to cause neonatal hypotonia and severe feeding problems. Failure to thrive usually persists for several years, followed by a period of hyperphagia and excessive weight gain. Growth retardation is usually observed, which responds well to growth hormone therapy. In addition, minor facial dysmorphism, nasal speech, viscous saliva, hypergonadotropic hypogonadism and learning problems are frequently observed. How PREPL deficiency causes these clinical manifestations remains unknown. PREPL is highly expressed in brain and based on gene coexpression network architecture it has been placed in a group enriched with markers of neurons and synaptic proteins. PREPL is predicted to be a serine oligopeptidase based on its homology with prolyl endopeptidase (PREP) and the presence of an active catalytic triad. However, until now no substrates have been found. Recent observations that PREP has non-catalytic functions in the cytoplasm through interactions with its amino- terminal propeller domain, suggests that of PREPL may also have biological functions independent of its predicted peptidase activity. This raises the possibility that PREP and PREPL are homologous, not just by name but also by nature.

Hyperhomocysteinemia is increasingly recognized as an independent risk factor for several cerebral, vascular, ocular, and agerelated disorders. Whether it is a cause or a consequence or a mere marker necessitates further clarification. This review focuses on the pathophysiological aspects of homocysteine's involvement in neurodegenerative and neuropsychiatric disorders and complications. The pharmacological agents (antiepileptic drugs, L-DOPA) augment the homocysteine levels, thus, raising concern for physicians. The mechanisms underlying the enhanced homocysteine levels and its related pathophysiological cascades remain poorly understood, inspite of numerous epidemiological and research studies that have been carried out in recent years. This article will review the current understanding of these underlying mechanisms and the research being carried with homocysteine as a core molecule.

Vascular dementia (VAD), the second most common form of dementia after Alzheimer's disease (AD) is characterized by a cognitive deficit of cerebrovascular origin. As for AD, the main proposed treatment is based on cholinesterase inhibitors. However, randomized clinical trials (RCTs) with cholinesterase inhibitors in VAD reported modest - though sometimes statistically significant - clinical efficacy. Non-cholinergic drugs with diverse rationales and mechanisms of action have also been tested in a few RCTs for VAD; the outcomes measured are variable and the evidence of efficacy is weak. The limitations of pharmacological treatment for VAD have prompted a different strategy, i.e. primary prevention aimed at reducing vascular risk factors. Several epidemiological studies reported associations of hypertension, type 2 diabetes, obesity, and inflammation with VAD and in some cases, AD. These all coincide with those of stroke, which in turn is an established factor for cognitive decline and VAD. Here too, only a few RCTs have looked at prevention of these factors, except hypertension. Some pharmacological classes are particularly promising from the clinical and experimental viewpoints: Ca2+ channel blockers and drugs affecting the renin-angiotensin system may act independently of the effects on blood pressure. Despite some conflicting results and the need for further work, the control of risk factors might prevent cognitive decline and VAD in the elderly. The benefit of tackling vascular factors is probably larger when also considering the prevention of stroke. The objective of this review is to analyze the pharmacological treatment and prevention of VAD and their outcome. The literature on Pubmed from 1980 to 2009 was examined.

Inflammatory signals generated within the brain and peripheral nervous system direct diverse biological processes. Key amongst the inflammatory molecules is tumor necrosis factor-alpha (TNF-α), a potent pro-inflammatory cytokine that, via binding to its associated receptors, is considered to be a master regulator of cellular cascades that control a number of diverse processes coupled to cell viability, gene expression, synaptic integrity and ion homeostasis. Whereas a self-limiting neuroinflammatory response generally results in the resolution of an intrinsically or extrinsically triggered insult by the elimination of toxic material or injured tissue to restore brain homeostasis and function, in the event of an unregulated reaction, where the immune response persists, inappropriate chronic neuroinflammation can ensue. Uncontrolled neuroinflammatory activity can induce cellular dysfunction and demise, and lead to a selfpropagating cascade of harmful pathogenic events. Such chronic neuroinflammation is a typical feature across a range of debilitating common neurodegenerative diseases, epitomized by Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis, in which TNF-α expression appears to be upregulated and may represent a valuable target for intervention. Elaboration of the protective homeostasis signaling cascades from the harmful pathogenic ones that likely drive disease onset and progression could aid in the clinical translation of approaches to lower brain and peripheral nervous system TNF-α levels, and amelioration of inappropriate neuroinflammation.

In the normal brain, cellular types that compose the neurovascular unit, including neurons, astrocytes and endothelial cells express pannexins and connexins, which are protein subunits of two families that form plasma membrane channels. Most available evidence in mammals indicated that endogenously expressed pannexins only form hemichannels, and connexins form both gap junction channels and hemichannels. While gap junction channels connect the cytoplasm of contacting cells and coordinate electrical and metabolic activities, hemichannels communicate intra- and extracellular compartments and serve as diffusional pathways for ions and small molecules. Here, evidence supporting the functional role of hemichannels in the neurovascular unit and white matter under physiological and pathological conditions are reviewed. A sub-threshold acute pathological threatening condition (e.g., stroke and brain infection) leads to glial cell activation, which maintains an active defense and restores the normal function of the neurovascular unit. However, if the stimulus is deleterious, microglia and the endothelium become overactivated, both releasing bioactive molecules (e.g., glutamate, cytokines, prostaglandins and ATP) that increase the activity of astroglial hemichannels, reducing the astrocyte neuroprotective functions, and further reducing neuronal cell viability. Moreover, ATP is known to contribute to myelin degeneration of axons. Consequently, hemichannels might play a relevant role in the excitotoxic response of oligodendrocytes observed in ischemia and encephalomyelitis. Regulated changes in hemichannel permeability in healthy brain cells can have positive consequences in terms of paracrine/autocrine signaling, whereas persistent changes in cells affected by neurological disorders can be detrimental. Therefore, blocking hemichannels expressed by glial cells and/or neurons of the inflamed central nervous system might prevent neurovascular unit dysfunction and neurodegeneration.