An Increasing Danger of Zoonotic Orthopoxvirus Infections

On May 8, 1980, the World Health Assembly at its 33^rd session solemnly declared that the world and all its peoples had won freedom from smallpox and recommended ceasing the vaccination of the population against smallpox. Currently, a larger part of the world population has no immunity not only against smallpox but also against other zoonotic orthopoxvirus infections. Recently, recorded outbreaks of orthopoxvirus diseases not only of domestic animals but also of humans have become more frequent. All this indicates a new situation in the ecology and evolution of zoonotic orthopoxviruses. Analysis of state-of-the-art data on the phylogenetic relationships, ecology, and host range of orthopoxviruses—etiological agents of smallpox (variola virus, VARV), monkeypox (MPXV), cowpox (CPXV), vaccinia (VACV), and camelpox (CMLV)—as well as the patterns of their evolution suggests that a VARV-like virus could emerge in the course of natural evolution of modern zoonotic orthopoxviruses. Thus, there is an insistent need for organization of the international control over the outbreaks of zoonotic orthopoxvirus infections in various countries to provide a rapid response and prevent them from developing into epidemics.The genus Orthopoxvirus of the family Poxviridae comprises the species variola (smallpox) virus (VARV), with human as its only sensitive host; zoonotic species monkeypox virus (MPXV), cowpox virus (CPXV), vaccinia virus (VACV), and camelpox virus (CMLV); and several others. These orthopoxviruses are immunologically cross-reactive and cross-protective, so that infection with any member of this genus provides protection against infection with any other member of the genus [1], [2]. Traditionally, the species of the Orthopoxvirus genus have been named primarily according to the host animal from which they were isolated and identified based on a range of biological characteristics [1]. Most frequently, zoonotic orthopoxviruses have been initially isolated from animals immediately close to humans being incidental hosts for the virus, the natural carriers of which are, as a rule, wild animals. Correspondingly, the name of an orthopoxvirus species does not reflect the actual animal that is its natural reservoir.With accumulation of the data on complete genome nucleotide sequences for various strains of orthopoxvirus species, it has been found that an interesting feature of the orthopoxvirus genomes is the presence of genes that are intact in one species but fragmented or deleted in another [3]–[8]. These data confirm the concept of a reductive evolution of orthopoxviruses, according to which the gene loss plays an important role in the evolutionary adaptation of progenitor virus to a particular environmental niche (host) and emergence of new virus species [9]. CPXV has the largest genome of all the modern representatives of the genus Orthopoxvirus, and this genome contains all the genes found in the other species of this genus [2], [4], [10]–[12]. Therefore, Cowpox virus was proposed as the closest of all the modern species to the progenitor virus for the genus Orthopoxvirus, while the remaining species, Variola virus included, had appeared as a result of multistage reductive evolution [4], [9], [13].VARV, the most pathogenic species for humans, has the smallest genome of all the orthopoxviruses [2]–[7]. This suggests a potential possibility for emergence of a VARV-like variant from the currently existing zoonotic orthopoxviruses with longer genomes in the course of natural evolution. It is known that although mutational changes are rather a rare event for the poxvirus DNA [13], characteristic of these viruses is the possibility of intermolecular and intramolecular recombinations, as well as genomic insertions and deletions [14], [15]. It has been recently found that duplication/amplification of genomic segments is typical of poxviruses, and in the case of a certain selective pressure (for example, host antiviral defenses), certain genes are able to relatively rapidly accumulate mutations that would provide the virus adaptation to new conditions, including a new host [16].The conducted analysis of the available archive data on smallpox and the history of ancient civilizations as well as the newest data on the evolutionary relationships of orthopoxviruses has allowed me to suggest the hypothesis that smallpox could have repeatedly emerged in the past via evolutionary changes of a zoonotic progenitor virus [17].Because of the cessation of the vaccination against smallpox after its eradication 35 years ago, a tremendous part of the world human population currently has no immunity not only against smallpox, but also against any other zoonotic orthopoxvirus infections. This new situation allows orthopoxviruses to circulate in the human population and, as a consequence, should alter several established concepts on the ecology and range of sensitive hosts for various orthopoxvirus species.The most intricate case is the origin of VACV. For many decades, VACV has been used for vaccinating humans against smallpox, and it was considered that this virus, variolae vaccinae, originates from zoonotic CPXV, introduced to immunization practice by Jenner as early as 1796 [1]. Only in the 20^th century was it found out that the orthopoxvirus strains used for smallpox vaccination significantly differ in their properties from both the natural CPXV isolates recovered from cows and the other orthopoxvirus species examined by that time [18]. Correspondingly, they were regarded as a separate species, Vaccinia virus [19]. Moreover, it was inferred that the VACV natural reservoir was unknown and numerous hypotheses attempted to explain the origin of this virus while passaging progenitor viruses in animals in the process of vaccine production [1], [2], [20].The issue of VACV origin was somewhat clarified after sequencing the complete genome of horsepox virus (HSPV) [21], which appeared to be closely related to the sequenced VACV strains. Only after this was attention paid to the fact that Jenner specified the origin of his vaccine from an infection of the heels of horses (“grease”) and indicated that the vaccine became more suitable for human use after passage through the cow [20]. This suggests that VACV may originate from a zoonotic HSPV, which naturally persisted concurrently with CPXV. Some facts suggest that the infectious materials not only from cow lesions but also from horse lesions were used for smallpox vaccination in the 19^th century. The vaccine lymph from the horse gave the most satisfactory results in inducing an anti-smallpox immunity as well as less side reactions [1]. By all accounts, they gradually commenced using HSPV isolates for smallpox vaccination, the future generations of which recovered decades later were ascribed to the separate species Vaccinia virus [19], rather than CPXV for smallpox vaccination everywhere.Since the 1960s, VACVs have been repeatedly isolated in Brazil [22]. The first VACV isolates were recovered from wild rodents (sentinel mice and rice rat) [23]. Since 1999, an ever-increasing number of exanthematous outbreaks affecting dairy cows and their handlers have been recorded [24]–[27], supplemented recently with outbreaks among horses [28], [29]. Several VACV strains have been isolated during these outbreaks from cows, horses, humans, and rodents [22], [27], [28], [30], [31]. The questions that arise are when and how VACV entered Brazil and the wild nature of the American continent. The more widespread point of view is that VACV strains could be transmitted from vaccinated humans to domestic animals and further to wild ones with subsequent adaptation to the rural environment [22]. My standpoint implies that HSPV/VACV could have been repeatedly accidentally imported from Europe to South America with the infected horses or rodents to be further introduced into wildlife. Possibly, the latter hypothesis more adequately reflects the actual pathway of VACV transmission to the Brazilian environment, since recent phylogenetic studies have suggested an independent origin for South American VACV isolates, distinct from the vaccine strains used on this continent during the WHO smallpox eradication campaign [22], [32]. Presumably, genome-wide sequencing of the viruses will give a more precise answer to the origin of VACV variants isolated in Brazil.In the past, the outbreaks of buffalopox had occurred frequently in various states of India as well as in Pakistan, Bangladesh, Indonesia, Egypt, and other countries [33]. The causative agent, buffalopox virus (BPXV), is closely related to VACV and affiliated with the species Vaccinia virus, genus Orthopoxvirus [2], [34]. Recently, mass outbreaks of buffalopox in domestic buffaloes along with severe zoonotic infection in milk attendants were recorded at various places in India [35], [36]. In several buffalopox outbreaks, the BPXV-caused infections were recorded in cows in the same herds [37]. An increase in BPXV transmission to different species, including buffaloes, cows, and humans, suggests the reemergence of zoonotic buffalopox infection [35], [38]. The buffalopox outbreaks recorded in different distant regions of India are likely to suggest the presence of an abundant natural BPXV reservoir represented by wild animals, most probably rodents. Correspondingly, it is of the paramount importance to perform a large-scale study of the presence of orthopoxviruses in wild animals of India.Thus, yet incomplete data on the modern ecology of VACV and BPXV allow for speculation that the orthopoxviruses belonging to the species Vaccinia virus have a wide host range, are zoonotic, are currently spread over large areas in Eurasia and South America, and that their natural carriers are several rodents.CPXV has relatively low pathogenicity for humans but has a wide range of sensitive animal hosts [2], [39]. Human cowpox is a rare sporadic disease, which develops when CPXV is transmitted from an infected animal to human [2], [40]. This disease is mainly recorded in Europe. In wildlife, CPXV carriers are asymptomatically infected rodents [41], [42]. During the last two decades, reports on an increasing number of CPXV infections in cats, rats, exotic animals, and humans have been published [43]–[47]. Comparative studies of the properties of CPXV isolates recovered from various hosts at different times and in several geographic zones have shown sufficient intraspecific variations [2], [48], [49]. A recent phylogenetic analysis of the complete genomes of 12 CPXV strains recovered from humans and several animal species suggests that they be split into two major Cowpox virus–like and Vaccinia virus–like clades [50]. This means that the criteria of the separation of orthopoxviruses into these two species should be corrected.MPXV is a zoonotic virus causing a human infection similar to smallpox in its clinical manifestations with a lethality rate of 1–8% [51]. The natural reservoir of MPXV is various species of African rodents [8], [10]. The active surveillance data in the same health zone (Democratic Republic of Congo) from the 1980s to 2006–2007 suggest a 20-fold increase in human monkeypox incidence 30 years after the cessation of the smallpox vaccination campaign [52]. This poses the question of whether MPXV can acquire the possibility of a high human-to-human transmission rate, characteristic of VARV, under conditions of a long-term absence of vaccination and considerably higher incidence of human infection. If this occurs, humankind will face a problem considerably more complex than with the smallpox eradication. First and foremost, this is determined by the fact that MPXV, unlike VARV, has its natural reservoir represented by numerous African rodents [2], [53].In its biological properties and according to the data of phylogenetic analysis of the complete virus genomic sequence, CMLV is closest to VARV, the causative agent of smallpox, as compared with the other orthopoxvirus species [1], [8]. Camelpox is recognized as one of the most important viral diseases in camels. This infection was first described in India in 1909. Subsequently, camelpox outbreaks have been reported in many countries of the Middle East, Asia, and Africa [54], [55]. Until recently, it has been commonly accepted that the host range of CMLV is confined to one animal species, camels [1], [55]. However, the first human cases of camelpox have been recently confirmed in India [56]. This suggests that camelpox could be a zoonotic disease. Since camelpox outbreaks occur irregularly in distant regions of the world and the viruses isolated during these outbreaks display different degrees of virulence [55], it is possible to postulate the presence of a wildlife animal reservoir of CMLV other than camels. Since the camelpox outbreaks are usually associated with the rainy season of the year, when rodents are actively reproducing, it is likely that rodents could be the natural carriers of CMLV.It is known that most of the emerging human pathogens originate from zoonotic pathogens [57]–[59]. Many viruses do not cause the disease in their natural reservoir hosts but can be highly pathogenic when transmitted to a new host species. Emerging and reemerging human pathogens more often are those with broad host ranges. The viruses able to infect many animal species are evolutionarily adapted to utilizing different cell mechanisms for their reproduction and, thus, can extend/change their host range with a higher probability [58].There are no fundamental prohibitions for the possible reemergence of smallpox or a similar human disease in the future as a result of natural evolution of the currently existing zoonotic orthopoxviruses. An ever-increasing sensitivity of the human population to zoonotic orthopoxviruses, resulting from cessation of the mass smallpox vaccination, elevates the probability for new variants of these viruses, potentially dangerous for humans, to emerge. However, the current situation is radically different from the ancient one, since many outbreaks of orthopoxvirus infections among domestic animals and humans are recorded and studied.Recently, the efforts of scientists under WHO control are directed to the development of state-of-the-art methods for VARV rapid identification as well as design of new generation safe smallpox vaccines and drugs against VARV and other orthopoxviruses [60]. The designed promising anti-orthopoxvirus drugs display no pronounced virus species specificity. Therefore, they are applicable in the outbreaks caused by any orthopoxvirus species. International acceptance of the designed highly efficient anti-orthopoxvirus drugs ST-246 and CMX001 [60] is of paramount importance.In the areas of high incidence of zoonotic orthopoxviral infections, it would be purposeful to vaccinate domestic and zoo animals as well as the persons closely associated with them using state-of-the-art safe vaccines based on VACV, which has a wide range of sensitive hosts. This would considerably decrease the likelihood for such infections to spread from wildlife into the human environment.In the African region endemic for monkeypox, which also displays a high rate of HIV infection, the population could be vaccinated with the VACV strain MVA, which has been recently demonstrated to be safe even for HIV-infected persons [61].Taking into account the above mentioned increased incidence of outbreaks of animal and human orthopoxvirus infections and their potential danger, it is important to accelerate organization of the international Smallpox Laboratory Network, discussed by the WHO Advisory Committee on Variola Virus Research [62], [63], and orient this network to express diagnosing not only of VARV but also of other zoonotic orthopoxviruses. This will provide constant monitoring of these infections in all parts of the world and make it possible to prevent the development of small outbreaks into expanded epidemics, thereby decreasing the risk of evolutional changes and emergence of an orthopoxvirus highly pathogenic for humans.The international system for clinical sampling and identification of infectious agents has been worked out and optimized while implementing the global smallpox eradication program under the aegis of the WHO as well as anti-epidemic measures and methods for mass vaccination [1]. The accumulated experience is of paramount importance for the establishment of international control not only over currently existing orthopoxvirus infections but also other emerging and reemerging diseases.     

Viral OTU Deubiquitinases: A Structural and Functional Comparison

Recent studies have revealed that proteases encoded by three very diverse RNA virus groups share structural similarity with enzymes of the Ovarian Tumor (OTU) superfamily of deubiquitinases (DUBs). The publication of the latest of these reports in quick succession prevented proper recognition and discussion of the shared features of these viral enzymes. Here we provide a brief structural and functional comparison of these virus-encoded OTU DUBs. Interestingly, although their shared structural features and substrate specificity tentatively place them within the same protease superfamily, they also show interesting differences that trigger speculation as to their origins.The covalent attachment of ubiquitin (Ub) to protein substrates, i.e., ubiquitination, plays a pivotal regulatory role in numerous cellular processes [1]–[5]. Ubiquitination can be reversed by deubiquitinases (DUBs) [6] and, not surprisingly, various virus groups encode such DUBs to influence ubiquitin-mediated host cell processes [7]–[21]. Some of these viral DUBs resemble proteases belonging to the Ovarian Tumor (OTU) superfamily [22]–[28]. Makarova et al. previously identified OTU proteases as a novel superfamily of cysteine proteases from different organisms [29], and their bioinformatics-based analysis included several of the viral enzymes discussed here. Recently reported structures of these viral DUBs include the OTU domains of the nairoviruses Crimean-Congo hemorrhagic fever virus (CCHFV) [22]–[24] and Dugbe virus (DUGV) [25], the papain-like protease (PLP2) domain of the arterivirus equine arteritis virus (EAV) [26], and the protease (PRO) domain of the tymovirus turnip yellow mosaic virus (TYMV) (Figure 1A–1D) [27], [28]. These viruses are strikingly diverse, considering that nairoviruses are mammalian negative-strand RNA viruses, while the mammalian arteriviruses and plant tymoviruses belong to separate orders of positive-strand RNA viruses.Open in a separate windowFigure 1Viral and eukaryotic OTU domain structures and viral protein context.Crystal structures of (A) CCHFV OTU (3PT2) [23], (B) DUGV OTU (4HXD) [25], (C) EAV PLP2 (4IUM) [26], (D) TYMV PRO (4A5U) [27], [28], (E) yeast OTU1 (3BY4) [57], and (F) human OTUD3 (4BOU) [46]. The β-hairpin motifs of CCHFV OTU and DUGV OTU are indicated in boxes in panels A and B, respectively, and the zinc-finger motif of EAV PLP2 is boxed in panel C. Active sites are indicated with arrows. The CCHFV OTU, DUGV OTU, EAV PLP2, and yeast OTU1 domains were crystallized in complex with Ub, which has been removed for clarity. Structure images were generated using PyMol [60]. (G) Schematic representation of the CCHFV large (L) protein [61], [62]. A similar organization is found in the DUGV L protein, but is not depicted. The OTU domain resides in the N-terminal region of this protein and is not involved in autoproteolytic cleavage events [48]. (H) Schematic representation of the EAV polyprotein 1ab [63]. PLP2 resides in nonstructural protein 2 (nsp2) and is responsible for the cleavage between nsp2 and nsp3 [51]. (I) Schematic representation of the TYMV ORF1 polyprotein [50]. PRO resides in the N-terminal product of this polyprotein and is responsible for two internal cleavages [49], [50]. Key replicative enzymes are indicated in G, H, and I. Colored arrowheads denote cleavage sites for the indicated protease domains. HEL, helicase; PLP, papain-like protease; RdRp, RNA-dependent RNA polymerase; SP, serine protease.Ubiquitination often involves the formation of polyubiquitin chains [1], which can target the ubiquitinated substrate to the proteasome for degradation [2] or modulate its protein–protein interactions, as in the activation of innate immune signaling pathways [3], [4]. Interestingly, several cellular OTU DUBs were found to negatively regulate innate immunity [30]–[33]. Likewise, both nairovirus OTU and arterivirus PLP2 were recently shown to inhibit innate immune responses by targeting ubiquitinated signaling factors [7]–[9], [26], [34], [35]. In contrast to eukaryotic OTU DUBs, both of these viral proteases were found to also deconjugate the Ub-like protein interferon-stimulated gene 15 (ISG15) [7], [36], which inhibits viral replication via a mechanism that is currently poorly understood [37]. Interestingly, coronaviruses (which, together with the arteriviruses, belong to the nidovirus order) also encode papain-like proteases targeting both Ub and ISG15 that were shown to inhibit innate immunity [11]–[13], [38]–[42] but belong to the ubiquitin-specific protease (USP) class of DUBs [6], [43], [44]. The presence of functionally similar, yet structurally different proteases in distantly related virus families highlights the potential benefits to the virus of harboring such enzymes.The proteasomal degradation pathway is an important cellular route to dispose of viral proteins, as exemplified by the turnover of the TYMV polymerase [45]. Moreover, the degradation of this protein is specifically counteracted by the deubiquitinase activity of TYMV PRO, which thus promotes virus replication [10]. The functional characterization of viral OTU DUBs remains incomplete and future studies will likely reveal additional roles in replication and virus–host interplay.Polyubiquitin chains can adopt a number of different configurations, depending on the type of covalent linkage present within the polymer [1]. A distal Ub molecule can be linked via its C-terminus to one of seven internal lysine residues present in a proximal Ub molecule via an isopeptide bond. Alternatively, in the case of linear chains, the C-terminus of the distal Ub is covalently linked to the N-terminal methionine residue of the proximal Ub via a peptide bond. While human OTU proteases often show a distinct preference for one or two isopeptide linkage types [46], nairovirus OTUs and TYMV PRO appear to be more promiscuous in their substrate preference [22], [25]. However, like most human OTU proteases, they seem unable to cleave linear polyubiquitin chains in vitro [22], [25], [46]. Arterivirus PLP2 has not been extensively studied in this respect.It is important to note that many positive-strand RNA viruses, including arteriviruses and tymoviruses, encode polyproteins that are post-translationally cleaved by internal protease domains [47]. Thus, while CCHFV OTU is not involved in viral protein cleavage and its activity seems dispensable for replication (Figure 1G) [48], both arterivirus PLP2 and tymovirus PRO are critically required for viral replication due to their primary role in polyprotein maturation (Figure 1H, 1I) [49]–[53]. Interestingly, while both EAV PLP2 and TYMV PRO can process peptide bonds in cis and in trans [50], [51], PRO does not cleave peptide bonds in linear polyubiquitin chains in vitro [25]. To date, activity of EAV PLP2 towards linear polyubiquitin chains has not been reported.Based on mutagenesis of putative catalytic residues, arterivirus PLP2 and tymovirus PRO were initially generally classified as papain-like cysteine proteases [51], [54], [55]. Now that crystal structures of these proteases are available, it is possible to search the DALI server [56] in order to identify structurally similar domains. Using the 3-dimensional coordinates of TYMV PRO, the most recently solved structure of a viral OTU protease, such a query identifies structural similarity with eukaryotic OTU DUBs as well as the nairovirus OTU domains and EAV PLP2 ([57] further highlights their similarities (Figure 2A–2C), and these comparisons together clearly position them within the OTU DUB superfamily. Sequence comparisons alone were insufficient to demonstrate this conclusively, as the similarity of viral OTU domains to each other and to eukaryotic OTU proteases is very limited and mostly restricted to the areas surrounding the active site residues [29].Open in a separate windowFigure 2Superpositions of the viral OTU proteases with yeast OTU1 and one another.Superpositions of yeast OTU1 (3BY4) [57] with (A) CCHFV OTU (3PT2) [23], RMSD: 1.8 Å over 112 residues, (B) EAV PLP2 (4IUM) [26], RMSD: 2.8 Å over 69 residues, and (C) TYMV PRO (4A5U) [27], [28], RMSD: 1.4 Å over 76 residues. Superpositions of the yeast OTU1-Ub complex with (D) the CCHFV OTU-Ub complex and (E) the EAV PLP2-Ub complex, highlighting the difference in the orientation of Ub between the two viral OTU domains versus the eukaryotic yeast OTU1 domain. The Ub that is complexed with yeast OTU1 is depicted in yellow, while the Ub complexed with CCHFV OTU or EAV PLP2 is depicted in orange. (F) Superposition of EAV PLP2 and TYMV PRO, RMSD: 2.5 Å over 53 residues. (G) Close-up of the active site region (boxed) of the superposition depicted in F. Side chains of the catalytic cysteine (Cys270 and Cys783 for EAV PLP2 and TYMV PRO, respectively) and histidine (His332 and His869 for EAV PLP2 and TYMV PRO, respectively) residues are shown as sticks, as well as the active site Asn263 for EAV PLP2. The backbone amide group of Asp267 likely contributes to the formation of the oxyanion hole in the active site of EAV PLP2, yet a functionally equivalent residue is absent in TYMV PRO. The Gly266 and Gly268 residues flanking Asp267 in EAV PLP2 are depicted as sticks as well, for clarity. Note the alternative orientation of the active site cysteine residue of TYMV PRO which, unlike EAV PLP2, was not determined in covalent complex with an Ub suicide substrate. All alignments were generated using the PDBeFOLD server [64], and thus the reported RMSD values differ from those reported in [60]. RMSD, root-mean-square deviation.

Table 1

Three-dimensional structural alignment of viral OTU domains against selected structures in the Protein Data Bank using the DALI server [56].

The Role of Genomics in Tracking the Evolution of Influenza A Virus

Influenza A virus causes annual epidemics and occasional pandemics of short-term respiratory infections associated with considerable morbidity and mortality. The pandemics occur when new human-transmissible viruses that have the major surface protein of influenza A viruses from other host species are introduced into the human population. Between such rare events, the evolution of influenza is shaped by antigenic drift: the accumulation of mutations that result in changes in exposed regions of the viral surface proteins. Antigenic drift makes the virus less susceptible to immediate neutralization by the immune system in individuals who have had a previous influenza infection or vaccination. A biannual reevaluation of the vaccine composition is essential to maintain its effectiveness due to this immune escape. The study of influenza genomes is key to this endeavor, increasing our understanding of antigenic drift and enhancing the accuracy of vaccine strain selection. Recent large-scale genome sequencing and antigenic typing has considerably improved our understanding of influenza evolution: epidemics around the globe are seeded from a reservoir in East-Southeast Asia with year-round prevalence of influenza viruses; antigenically similar strains predominate in epidemics worldwide for several years before being replaced by a new antigenic cluster of strains. Future in-depth studies of the influenza reservoir, along with large-scale data mining of genomic resources and the integration of epidemiological, genomic, and antigenic data, should enhance our understanding of antigenic drift and improve the detection and control of antigenically novel emerging strains.Influenza is a single-stranded, negative-sense RNA virus that causes acute respiratory illness in humans. In temperate regions, winter influenza epidemics result in 250,000–500,000 deaths per year; in tropical regions, the burden is similar [1],[2]. Influenza viruses of three genera or types (A, B, and C) circulate in the human population. Influenza viruses of the types B and C evolve slowly and circulate at low levels. Type A evolves rapidly and can evade neutralization by antibodies in individuals who have been previously infected with, or vaccinated against, the virus. As a result it regularly causes large epidemics. Furthermore, distinct reservoirs of influenza A exist in other mammals and in birds. Four times in the last hundred years these reservoirs have provided genetic material for novel viruses that have caused global pandemics [3]–[8].The genome of influenza A viruses comprises eight RNA segments of 0.9–2.3 kb that together span approximately 13.5 kb and encode 11 proteins [9]. Segment 4 encodes the major surface glycoprotein called hemagglutinin (H), which is responsible for attaching the virus to sialic acid residues on the host cell surface and fusing the virus membrane envelope with the host cell membrane, thus delivering the viral genome into the cell (Figure 1). Segment 6 encodes another surface glycoprotein called neuraminidase (N), which cleaves terminal sialic acid residues from glycoproteins and glycolipids on the host cell surface, thus releasing budding viral particles from an infected cell [10]. Influenza A viruses are further classified into distinct subtypes based on the genetic and antigenic characteristics of these two surface glycoproteins. Sixteen hemagglutinin (H1–16) and nine neuraminidase subtypes (N1–9) are known to exist, and they occur in various combinations in influenza viruses endemic in aquatic birds [10],[11]. Viruses with the subtype composition H1N1 and H3N2 have been circulating in the human population for several decades. Of these two subtypes, H3N2 evolves more rapidly, and has until recently caused the majority of infections [1],[12],[13]. In the spring of 2009, however, a new H1N1 virus originating from swine influenza A viruses, and only distantly related to the H1N1 already circulating, gained hold in the human population. The emergence of this virus has initiated the first influenza pandemic of the twenty-first century [7],[14],[15].Open in a separate windowFigure 1Schematic representation of an influenza A virion.Three proteins, hemagglutinin (HA, a trimer of three identical subunits), neuraminidase (NA, a tetramer of four identical subunits), and the M2 transmembrane proton channel (a tetramer of four identical subunits), are anchored in the viral membrane, which is composed of a lipid bilayer. The large, external domains of hemagglutinin and neuraminidase are the major targets for neutralizing antibodies of the host immune response. The M1 matrix protein is located below the membrane. The genome of the influenza A virus is composed of eight individual RNA segments (conventionally ordered by decreasing length, bottom row), which each encode one or two proteins. Inside the virion, the eight RNA segments are packaged in a complex with nucleoprotein (NP) and the viral polymerase complex, consisting of the PA, PB1, and PB2 proteins.Hemagglutinin is about five times more abundant than neuraminidase in the viral membrane and is the major target of the host immune response [16]–[18]. Following exposure to the virus, whether by infection or vaccination, the host immune system acquires the capacity to produce neutralizing antibodies against the viral surface glycoproteins. These antibodies participate in clearing an infection and may protect an individual from future infections for many decades [19]. Five exposed regions on the surface of hemagglutinin, called epitope sites, are predominantly recognized by such antibodies [16],[17]. However, the human subtypes of influenza A continuously evolve and acquire genetic mutations that result in amino acid changes in the epitopes. These changes reduce the protective effect of antibodies raised against previously circulating viral variants. This “antigenic drift” necessitates frequent modification and readministration of the influenza vaccine to ensure efficient protection (Box 1).

Box 1. Broadly Protective Vaccines

Current influenza vaccines are based on detergent-inactivated viruses. They elicit antibodies with a narrow range of protection that target predominantly the variable regions of the hemagglutinin protein. Accordingly, the seasonal influenza vaccine includes one strain with segments of the surface proteins for each of the A/H1N1, A/H3N2 and B viruses, and it is updated every 1–3 years to match the predominant variants of influenza. Research into vaccines that offer broader protection across diverse subtypes and antigenic drift variants is ongoing [21], [59]–[61]. This research is particularly important with respect to the emergence of novel viruses with pandemic potential, such as the 2009 H1N1 virus. In such an event, the time period between the detection of the virus and the onset of a pandemic is too short to produce a specific vaccine for immediate vaccination of the population. Work in this area is focused on developing vaccines that elicit antibodies against conserved viral components, such as certain regions of hemagglutinin, neuraminidase, and the M2 proton channel in the viral membrane [60]. Other types of vaccines based on live attenuated viruses or plasmid DNA expression vectors, or supplemented with adjuvants, show promise in inducing a more broadly protective immune response [61].To monitor for novel emerging strains, the World Health Organization (WHO) maintains a global surveillance program. A panel of experts meets twice a year to review antigenic, genetic, and epidemiological data and decides on the vaccine composition for the next winter season in the northern or southern hemisphere [20]. If an emerging antigenic variant is detected and judged likely to become predominant, an update of the vaccine strain is recommended. This “predict and produce” approach mostly results in efficient vaccines that substantially limit the morbidity and mortality of seasonal epidemics [21]. The recommendation has to be made almost a year before the season in which the vaccine is used, however, because of the time required to produce and distribute a new vaccine. Problems arise when an emerging variant is not identified early enough for an update of the vaccine composition [22]–[24]. Thus, gaining a detailed understanding of the evolution and epidemiology of the virus is of the utmost importance, as it may lead to earlier identification of novel emerging variants [20].The development of high-throughput sequencing has recently provided large datasets of high-quality, complete genome sequences for viral isolates collected in a relatively unbiased manner, regardless of virulence or other unusual characteristics [9],[25]. Analyses of the genome sequence data combined with large-scale antigenic typing [26],[27] have given insights into the pattern of global spread, the genetic diversity during seasonal epidemics, and the dynamics of subtype evolution. Influenza data repositories such as the NCBI Influenza Virus Resource (http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html) [28] and the Global Initiative on Sharing All Influenza Data (GISAID; http://platform.gisaid.org/) database [29] make the genomic information publicly available, together with epidemiological data for the sequenced isolates. The GISAID model for data sharing requires users to agree to collaborate with, and appropriately credit, all data contributors. A notable success of this initiative has been the contribution of countries, such as Indonesia and China, which have previously been reticent about placing data in the public domain. The WHO also supports the endeavor of rapid publication of all available sequences for influenza viruses and there is hope that comprehensive submission to public databases will soon become a reality [24],[30]. In the future, mining these resources and establishing a statistical framework based on epidemiological, antigenic, and genetic information could provide further insights into the rules that govern the emergence and establishment of antigenically novel variants and improve the potential for influenza prevention and control.     

The Cytoskeletal Protein ��-Actinin Regulates Acid-sensing Ion Channel 1a through a C-terminal Interaction

The acid-sensing ion channel 1a (ASIC1a) is widely expressed in central and peripheral neurons where it generates transient cation currents when extracellular pH falls. ASIC1a confers pH-dependent modulation on postsynaptic dendritic spines and has critical effects in neurological diseases associated with a reduced pH. However, knowledge of the proteins that interact with ASIC1a and influence its function is limited. Here, we show that α-actinin, which links membrane proteins to the actin cytoskeleton, associates with ASIC1a in brain and in cultured cells. The interaction depended on an α-actinin-binding site in the ASIC1a C terminus that was specific for ASIC1a versus other ASICs and for α-actinin-1 and -4. Co-expressing α-actinin-4 altered ASIC1a current density, pH sensitivity, desensitization rate, and recovery from desensitization. Moreover, reducing α-actinin expression altered acid-activated currents in hippocampal neurons. These findings suggest that α-actinins may link ASIC1a to a macromolecular complex in the postsynaptic membrane where it regulates ASIC1a activity.Acid-sensing ion channels (ASICs)² are H⁺-gated members of the DEG/ENaC family (1–3). Members of this family contain cytosolic N and C termini, two transmembrane domains, and a large cysteine-rich extracellular domain. ASIC subunits combine as homo- or heterotrimers to form cation channels that are widely expressed in the central and peripheral nervous systems (1–4). In mammals, four genes encode ASICs, and two subunits, ASIC1 and ASIC2, have two splice forms, a and b. Central nervous system neurons express ASIC1a, ASIC2a, and ASIC2b (5–7). Homomeric ASIC1a channels are activated when extracellular pH drops below 7.2, and half-maximal activation occurs at pH 6.5–6.8 (8–10). These channels desensitize in the continued presence of a low extracellular pH, and they can conduct Ca²⁺ (9, 11–13). ASIC1a is required for acid-evoked currents in central nervous system neurons; disrupting the gene encoding ASIC1a eliminates H⁺-gated currents unless extracellular pH is reduced below pH 5.0 (5, 7).Previous studies found ASIC1a enriched in synaptosomal membrane fractions and present in dendritic spines, the site of excitatory synapses (5, 14, 15). Consistent with this localization, ASIC1a null mice manifested deficits in hippocampal long term potentiation, learning, and memory, which suggested that ASIC1a is required for normal synaptic plasticity (5, 16). ASICs might be activated during neurotransmission when synaptic vesicles empty their acidic contents into the synaptic cleft or when neuronal activity lowers extracellular pH (17–19). Ion channels, including those at the synapse often interact with multiple proteins in a macromolecular complex that incorporates regulators of their function (20, 21). For ASIC1a, only a few interacting proteins have been identified. Earlier work indicated that ASIC1a interacts with another postsynaptic scaffolding protein, PICK1 (15, 22, 23). ASIC1a also has been reported to interact with annexin II light chain p11 through its cytosolic N terminus to increase cell surface expression (24) and with Ca²⁺/calmodulin-dependent protein kinase II to phosphorylate the channel (25). However, whether ASIC1a interacts with additional proteins and with the cytoskeleton remain unknown. Moreover, it is not known whether such interactions alter ASIC1a function.In analyzing the ASIC1a amino acid sequence, we identified cytosolic residues that might bind α-actinins. α-Actinins cluster membrane proteins and signaling molecules into macromolecular complexes and link membrane proteins to the actincytoskeleton (for review, Ref. 26). Four genes encode α-actinin-1, -2, -3, and -4 isoforms. α-Actinins contain an N-terminal head domain that binds F-actin, a C-terminal region containing two EF-hand motifs, and a central rod domain containing four spectrin-like motifs (26–28). The C-terminal portion of the rod segment appears to be crucial for binding to membrane proteins. The α-actinins assemble into antiparallel homodimers through interactions in their rod domain. α-Actinins-1, -2, and -4 are enriched in dendritic spines, concentrating at the postsynaptic membrane (29–35). In the postsynaptic membrane of excitatory synapses, α-actinin connects the NMDA receptor to the actin cytoskeleton, and this interaction is key for Ca²⁺-dependent inhibition of NMDA receptors (36–38). α-Actinins can also regulate the membrane trafficking and function of several cation channels, including L-type Ca²⁺ channels, K⁺ channels, and TRP channels (39–41).To better understand the function of ASIC1a channels in macromolecular complexes, we asked if ASIC1a associates with α-actinins. We were interested in the α-actinins because they and ASIC1a, both, are present in dendritic spines, ASIC1a contains a potential α-actinin binding sequence, and the related epithelial Na⁺ channel (ENaC) interacts with the cytoskeleton (42, 43). Therefore, we hypothesized that α-actinin interacts structurally and functionally with ASIC1a.     

Structural and Mechanistic Insights into Lunatic Fringe from a Kinetic Analysis of Enzyme Mutants

The Notch receptor is critical for proper development where it orchestrates numerous cell fate decisions. The Fringe family of β1,3-N-acetylglucosaminyltransferases are regulators of this pathway. Fringe enzymes add N-acetylglucosamine to O-linked fucose on the epidermal growth factor repeats of Notch. Here we have analyzed the reaction catalyzed by Lunatic Fringe (Lfng) in detail. A mutagenesis strategy for Lfng was guided by a multiple sequence alignment of Fringe proteins and solutions from docking an epidermal growth factor-like O-fucose acceptor substrate onto a homology model of Lfng. We targeted three main areas as follows: residues that could help resolve where the fucose binds, residues in two conserved loops not observed in the published structure of Manic Fringe, and residues predicted to be involved in UDP-N-acetylglucosamine (UDP-GlcNAc) donor specificity. We utilized a kinetic analysis of mutant enzyme activity toward the small molecule acceptor substrate 4-nitrophenyl-α-l-fucopyranoside to judge their effect on Lfng activity. Our results support the positioning of O-fucose in a specific orientation to the catalytic residue. We also found evidence that one loop closes off the active site coincident with, or subsequent to, substrate binding. We propose a mechanism whereby the ordering of this short loop may alter the conformation of the catalytic aspartate. Finally, we identify several residues near the UDP-GlcNAc-binding site, which are specifically permissive toward UDP-GlcNAc utilization.Defects in Notch signaling have been implicated in numerous human diseases, including multiple sclerosis (1), several forms of cancer (2-4), cerebral autosomal dominant arteriopathy with sub-cortical infarcts and leukoencephalopathy (5), and spondylocostal dysostosis (SCD)³ (6-8). The transmembrane Notch signaling receptor is activated by members of the DSL (Delta, Serrate, Lag2) family of ligands (9, 10). In the endoplasmic reticulum, O-linked fucose glycans are added to the epidermal growth factor-like (EGF) repeats of the Notch extracellular domain by protein O-fucosyltransferase 1 (11-13). These O-fucose monosaccharides can be elongated in the Golgi apparatus by three highly conserved β1,3-N-acetylglucosaminyltransferases of the Fringe family (Lunatic (Lfng), Manic (Mfng), and Radical Fringe (Rfng) in mammals) (14-16). The formation of this GlcNAc-β1,3-Fuc-α1, O-serine/threonine disaccharide is necessary and sufficient for subsequent elongation to a tetrasaccharide (15, 19), although elongation past the disaccharide in Drosophila is not yet clear (20, 21). Elongation of O-fucose by Fringe is known to potentiate Notch signaling from Delta ligands and inhibit signaling from Serrate ligands (22). Delta ligands are termed Delta-like (Delta-like1, -2, and -4) in mammals, and the homologs of Serrate are known as Jagged (Jagged1 and -2) in mammals. The effects of Fringe on Drosophila Notch can be recapitulated in Notch ligand in vitro binding assays using purified components, suggesting that the elongation of O-fucose by Fringe alters the binding of Notch to its ligands (21). Although Fringe also appears to alter Notch-ligand interactions in mammals, the effects of elongation of the glycan past the O-fucose monosaccharide is more complicated and appears to be cell type-, receptor-, and ligand-dependent (for a recent review see Ref. 23).The Fringe enzymes catalyze the transfer of GlcNAc from the donor substrate UDP-α-GlcNAc to the acceptor fucose, forming the GlcNAc-β1,3-Fuc disaccharide (14-16). They belong to the GT-A-fold of inverting glycosyltransferases, which includes N-acetylglucosaminyltransferase I and β1,4-galactosyltransferase I (17, 18). The mechanism is presumed to proceed through the abstraction of a proton from the acceptor substrate by a catalytic base (Asp or Glu) in the active site. This creates a nucleophile that attacks the anomeric carbon of the nucleotide-sugar donor, inverting its configuration from α (on the nucleotide sugar) to β (in the product) (24, 25). The enzyme then releases the acceptor substrate modified with a disaccharide and UDP. The Mfng structure (26) leaves little doubt as to the identity of the catalytic residue, which in all likelihood is aspartate 289 in mouse Lfng (we will use numbering for mouse Lunatic Fringe throughout, unless otherwise stated). The structure of Mfng with UDP-GlcNAc soaked into the crystals (26) showed density only for the UDP portion of the nucleotide-sugar donor and no density for two loops flanking either side of the active site. The presence of flexible loops that become ordered upon substrate binding is a common observation with glycosyltransferases in the GT-A fold family (18, 25). Density for the entire donor was observed in the structure of rabbit N-acetylglucosaminyltransferase I (27). In this case, ordering of a previously disordered loop upon UDP-GlcNAc binding may have contributed to increased stability of the donor. In the case of bovine β1,4-galactosyltransferase I, a section of flexible random coil from the apo-structure was observed to change its conformation to α-helical upon donor substrate binding (28). Both loops in Lfng are highly conserved, and we have mutated a number of residues in each to test the hypothesis that they interact with the substrates. The mutagenesis strategy was also guided by docking of an EGF-O-fucose acceptor substrate into the active site of the Lfng model as well as comparison of the Lfng model with a homology model of the β1,3-glucosyltransferase (β3GlcT) that modifies O-fucose on thrombospondin type 1 repeats (29, 30). The β3GlcT is predicted to be a GT-A fold enzyme related to the Fringe family (17, 18, 29).     

Sequential protein kinase C (PKC)-dependent and PKC-independent protein kinase D catalytic activation via Gq-coupled receptors: differential regulation of activation loop Ser(744) and Ser(748) phosphorylation

Protein kinase D (PKD) is a serine/threonine protein kinase rapidly activated by G protein-coupled receptor (GPCR) agonists via a protein kinase C (PKC)-dependent pathway. Recently, PKD has been implicated in the regulation of long term cellular activities, but little is known about the mechanism(s) of sustained PKD activation. Here, we show that cell treatment with the preferential PKC inhibitors GF 109203X or Gö 6983 blocked rapid (1–5-min) PKD activation induced by bombesin stimulation, but this inhibition was greatly diminished at later times of bombesin stimulation (e.g. 45 min). These results imply that GPCR-induced PKD activation is mediated by early PKC-dependent and late PKC-independent mechanisms. Western blot analysis with site-specific antibodies that detect the phosphorylated state of the activation loop residues Ser⁷⁴⁴ and Ser⁷⁴⁸ revealed striking PKC-independent phosphorylation of Ser⁷⁴⁸ as well as Ser⁷⁴⁴ phosphorylation that remained predominantly but not completely PKC-dependent at later times of bombesin or vasopressin stimulation (20–90 min). To determine the mechanisms involved, we examined activation loop phosphorylation in a set of PKD mutants, including kinase-deficient, constitutively activated, and PKD forms in which the activation loop residues were substituted for alanine. Our results show that PKC-dependent phosphorylation of the activation loop Ser⁷⁴⁴ and Ser⁷⁴⁸ is the primary mechanism involved in early phase PKD activation, whereas PKD autophosphorylation on Ser⁷⁴⁸ is a major mechanism contributing to the late phase of PKD activation occurring in cells stimulated by GPCR agonists. The present studies identify a novel mechanism induced by GPCR activation that leads to late, PKC-independent PKD activation.A rapid increase in the synthesis of lipid-derived second messengers with subsequent activation of protein phosphorylation cascades has emerged as a fundamental signal transduction mechanism triggered by multiple extracellular stimuli, including hormones, neurotransmitters, chemokines, and growth factors (1). Many of these agonists bind to G protein-coupled receptors (GPCRs),⁴ activate heterotrimeric G proteins and stimulate isoforms of the phospholipase C family, including β, γ, δ, and ε (reviewed in Refs. 1 and 2). Activated phospholipase Cs catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce the second messengers inositol 1,4,5-trisphosphate and diacylglycerol (DAG). Inositol 1,4,5-trisphosphate mobilizes Ca²⁺ from intracellular stores (3, 4) whereas DAG directly activates the classic (α, β, and γ) and novel (δ, ε, η, and θ) isoforms of PKC (5–7). Although it is increasingly recognized that each PKC isozyme has specific functions in vivo (5–8), the mechanisms by which PKC-mediated signals are propagated to critical downstream targets remain incompletely defined.PKD, also known initially as PKCμ (9, 10), and two recently identified serine protein kinases termed PKD2 (11) and PKCν/PKD3 (12, 13), which are similar in overall structure and primary amino acid sequence to PKD (14), constitute a new protein kinase family within the Ca²⁺/calmodulin-dependent protein kinase group (15) and separate from the previously identified PKCs (14). Salient features of PKD structure include an N-terminal regulatory region containing a tandem repeat of cysteine-rich zinc finger-like motifs (termed the cysteine-rich domain) that confers high affinity binding to phorbol esters and DAG (9, 16, 17), followed by a pleckstrin homology (PH) domain that negatively regulates catalytic activity (18, 19). The C-terminal region of the PKDs contains its catalytic domain, which is distantly related to Ca²⁺-regulated kinases.In unstimulated cells, PKD is in a state of low kinase catalytic activity maintained by the N-terminal domain, which represses the catalytic activity of the enzyme by autoinhibition. Consistent with this model, deletions or single amino acid substitutions in the PH domain result in constitutive kinase activity (18–20). Physiological activation of PKD within cells occurs via a phosphorylation-dependent mechanism first identified in our laboratory (21). In response to cellular stimuli, PKD is converted from a low activity form into a persistently active form that is retained during isolation from cells, as shown by in vitro kinase assays performed in the absence of lipid co-activators (21, 22). PKD activation has been demonstrated in response to engagement of specific GPCRs either by regulatory peptides (23–30) or lysophosphatidic acid (27, 31, 32); signaling through G_q, G₁₂, G_i, and Rho (27, 31–34); activation of receptor tyrosine kinases, such as the platelet-derived growth factor receptor (23, 35, 36); cross-linking of B-cell receptor and T-cell receptor in B and T lymphocytes, respectively (37–40); and oxidative stress (41–44).Throughout these studies, multiple lines of evidence indicated that PKC activity is necessary for rapid PKD activation within intact cells. For example, rapid PKD activation was selectively and potently blocked by cell treatment with preferential PKC inhibitors (e.g. GF 109203X or Gö 6983) that do not directly inhibit PKD catalytic activity (21, 22), implying that PKD activation in intact cells is mediated, directly or indirectly, through PKCs. In line with this conclusion, cotransfection of PKD with active mutant forms of “novel” PKCs (PKCs δ, ε, η, and θ) resulted in robust PKD activation in the absence of cell stimulation (21, 44–46). Many reports demonstrated the operation of a rapid PKC/PKD signaling cascade in response to multiple GPCR agonists in a broad range of cell types, including normal and cancer cells (reviewed in Ref. 14). Our previous studies identified Ser⁷⁴⁴ and Ser⁷⁴⁸ in the PKD activation loop (also referred as the activation segment or T-loop) as phosphorylation sites critical for PKC-mediated PKD activation (reviewed in Ref. 14). Collectively, these findings demonstrated the existence of rapidly activated PKC-PKD protein kinase cascade(s) and raised the possibility that some PKC-dependent biological responses involve PKD acting as a downstream effector.PKD has been reported recently to mediate several important cellular activities and processes, including signal transduction (30, 47–49), chromatin modification (50), Golgi organization and function (51, 52), c-Jun function (47, 53, 54), NFκB-mediated gene expression (43, 55, 56), and cell survival, migration, and differentiation and DNA synthesis and proliferation (reviewed in Ref. 14). Thus, mounting evidence indicates that PKD has a remarkable diversity of both its signal generation and distribution and its potential for complex regulatory interactions with multiple downstream pathways, leading to multiple responses, including long term cellular events. Despite increasing recognition of its importance, very little is known about the mechanism(s) of sustained PKD activation as opposed to the well documented rapid, PKC-dependent PKD activation.The results presented here demonstrate that prolonged GPCR-induced PKD activation is mediated by sequential PKC-dependent and PKC-independent phases of regulation. We report here, for the first time, that PKD autophosphorylation on Ser⁷⁴⁸ is a major mechanism contributing to the late phase of PKD activation occurring in cells stimulated by GPCR agonists. The present studies expand previous models of PKD regulation by identifying a novel mechanism induced by GPCR activation that leads to late, PKC-independent PKD activation.     

Intracellular Trafficking of Presenilin 1 Is Regulated by ��-Amyloid Precursor Protein and Phospholipase D1

Excessive accumulation of β-amyloid peptides in the brain is a major cause for the pathogenesis of Alzheimer disease. β-Amyloid is derived from β-amyloid precursor protein (APP) through sequential cleavages by β- and γ-secretases, whose enzymatic activities are tightly controlled by subcellular localization. Delineation of how intracellular trafficking of these secretases and APP is regulated is important for understanding Alzheimer disease pathogenesis. Although APP trafficking is regulated by multiple factors including presenilin 1 (PS1), a major component of the γ-secretase complex, and phospholipase D1 (PLD1), a phospholipid-modifying enzyme, regulation of intracellular trafficking of PS1/γ-secretase and β-secretase is less clear. Here we demonstrate that APP can reciprocally regulate PS1 trafficking; APP deficiency results in faster transport of PS1 from the trans-Golgi network to the cell surface and increased steady state levels of PS1 at the cell surface, which can be reversed by restoring APP levels. Restoration of APP in APP-deficient cells also reduces steady state levels of other γ-secretase components (nicastrin, APH-1, and PEN-2) and the cleavage of Notch by PS1/γ-secretase that is more highly correlated with cell surface levels of PS1 than with APP overexpression levels, supporting the notion that Notch is mainly cleaved at the cell surface. In contrast, intracellular trafficking of β-secretase (BACE1) is not regulated by APP. Moreover, we find that PLD1 also regulates PS1 trafficking and that PLD1 overexpression promotes cell surface accumulation of PS1 in an APP-independent manner. Our results clearly elucidate a physiological function of APP in regulating protein trafficking and suggest that intracellular trafficking of PS1/γ-secretase is regulated by multiple factors, including APP and PLD1.An important pathological hallmark of Alzheimer disease (AD)⁴ is the formation of senile plaques in the brains of patients. The major components of those plaques are β-amyloid peptides (Aβ), whose accumulation triggers a cascade of neurodegenerative steps ending in formation of senile plaques and intraneuronal fibrillary tangles with subsequent neuronal loss in susceptible brain regions (1, 2). Aβ is proteolytically derived from the β-amyloid precursor protein (APP) through sequential cleavages by β-secretase (BACE1), a novel membrane-bound aspartyl protease (3, 4), and by γ-secretase, a high molecular weight complex consisting of at least four components: presenilin (PS), nicastrin (NCT), anterior pharynx-defective-1 (APH-1), and presenilin enhancer-2 (PEN-2) (5, 6). APP is a type I transmembrane protein belonging to a protein family that includes APP-like protein 1 (APLP1) and 2 (APLP2) in mammals (7, 8). Full-length APP is synthesized in the endoplasmic reticulum (ER) and transported through the Golgi apparatus. Most secreted Aβ peptides are generated within the trans-Golgi network (TGN), also the major site of steady state APP in neurons (9–11). APP can be transported to the cell surface in TGN-derived secretory vesicles if not proteolyzed to Aβ or an intermediate metabolite. At the cell surface APP is either cleaved by α-secretase to produce soluble sAPPα (12) or reinternalized for endosomal/lysosomal degradation (13, 14). Aβ may also be generated in endosomal/lysosomal compartments (15, 16). In contrast to neurotoxic Aβ peptides, sAPPα possesses neuroprotective potential (17, 18). Thus, the subcellular distribution of APP and proteases that process it directly affect the ratio of sAPPα to Aβ, making delineation of the mechanisms responsible for regulating trafficking of all of these proteins relevant to AD pathogenesis.Presenilin (PS) is a critical component of the γ-secretase. Of the two mammalian PS gene homologues, PS1 and PS2, PS1 encodes the major form (PS1) in active γ-secretase (19, 20). Nascent PSs undergo endoproteolytic cleavage to generate an amino-terminal fragment (NTF) and a carboxyl-terminal fragment (CTF) to form a functional PS heterodimer (21). Based on observations that PSs possess two highly conserved aspartate residues indispensable for γ-secretase activity and that specific transition state analogue γ-secretase inhibitors bind to PS1 NTF/CTF heterodimers (5, 22), PSs are believed to be the catalytic component of the γ-secretase complex. PS assembles with three other components, NCT, APH-1, and PEN-2, to form the functional γ-secretase (5, 6). Strong evidence suggests that PS1/γ-secretase resides principally in the ER, early Golgi, TGN, endocytic and intermediate compartments, most of which (except the TGN) are not major subcellular sites for APP (23, 24). In addition to generating Aβ and cleaving APP to release the APP intracellular domain, PS1/γ-secretase cleaves other substrates such as Notch (25), cadherin (26), ErbB4 (27), and CD44 (28), releasing their respective intracellular domains. Interestingly, PS1/γ-secretase cleavage of different substrates seems to occur at different subcellular compartments; APP is mainly cleaved at the TGN and early endosome domains, whereas Notch is predominantly cleaved at the cell surface (9, 11, 29). Thus, perturbing intracellular trafficking of PS1/γ-secretase may alter interactions between PS1/γ-secretase and APP, contributing to either abnormal Aβ generation and AD pathogenesis or decreased access of PS1/γ-secretase to APP such that Aβ production is reduced. However, mechanisms regulating PS1/γ-secretase trafficking warrant further investigation.In addition to participating in γ-secretase activity, PS1 regulates intracellular trafficking of several membrane proteins, including other γ-secretase components (nicastrin, APH-1, and PEN-2) and the substrate APP (reviewed in Ref. 30). Intracellular APP trafficking is highly regulated and requires other factors such as mint family members and SorLA (2). Moreover, we recently found that phospholipase D1 (PLD1), a phospholipid-modifying enzyme that regulates membrane trafficking events, can interact with PS1, and can regulate budding of APP-containing vesicles from the TGN and delivery of APP to the cell surface (31, 32). Interestingly, Kamal et al. (33) identified an axonal membrane compartment that contains APP, BACE1, and PS1 and showed that fast anterograde axonal transport of this compartment is mediated by APP and kinesin-I, implying a traffic-regulating role for APP. Increased APP expression is also shown to decrease retrograde axonal transport of nerve growth factor (34). However, whether APP indeed regulates intracellular trafficking of proteins including BACE1 and PS1/γ-secretase requires further validation. In the present study we demonstrate that intracellular trafficking of PS1, as well as that of other γ-secretase components, but not BACE1, is regulated by APP. APP deficiency promotes cell surface delivery of PS1/γ-secretase complex and facilitates PS1/γ-secretase-mediated Notch cleavage. In addition, we find that PLD1 also regulates intracellular trafficking of PS1 through a different mechanism and more potently than APP.     

Polo-like Kinase 2 (PLK2) Phosphorylates ��-Synuclein at Serine 129 in Central Nervous System

Several neurological diseases, including Parkinson disease and dementia with Lewy bodies, are characterized by the accumulation of α-synuclein phosphorylated at Ser-129 (p-Ser-129). The kinase or kinases responsible for this phosphorylation have been the subject of intense investigation. Here we submit evidence that polo-like kinase 2 (PLK2, also known as serum-inducible kinase or SNK) is a principle contributor to α-synuclein phosphorylation at Ser-129 in neurons. PLK2 directly phosphorylates α-synuclein at Ser-129 in an in vitro biochemical assay. Inhibitors of PLK kinases inhibited α-synuclein phosphorylation both in primary cortical cell cultures and in mouse brain in vivo. Finally, specific knockdown of PLK2 expression by transduction with short hairpin RNA constructs or by knock-out of the plk2 gene reduced p-Ser-129 levels. These results indicate that PLK2 plays a critical role in α-synuclein phosphorylation in central nervous system.The importance of α-synuclein to the pathogenesis of Parkinson disease (PD)⁴ and the related disorder, dementia with Lewy bodies (DLB), is suggested by its association with Lewy bodies and Lewy neurites, the inclusions that characterize these diseases (1–3), and demonstrated by the existence of mutations that cause syndromes mimicking sporadic PD and DLB (4–6). Furthermore, three separate mutations cause early onset forms of PD and DLB. It is particularly telling that duplications or triplications of the gene (7–9), which increase levels of α-synuclein with no alteration in sequence, also cause PD or DLB.α-Synuclein has been reported to be phosphorylated on serine residues, at Ser-87 and Ser-129 (10), although to date only the Ser-129 phosphorylation has been identified in the central nervous system (11, 12). Phosphorylation at tyrosine residues has been observed by some investigators (13, 14) but not by others (10–12). Phosphorylation at Ser-129 (p-Ser-129) is of particular interest because the majority of synuclein in Lewy bodies contains this modification (15). In addition, p-Ser-129 was found to be the most extensive and consistent modification in a survey of synuclein in Lewy bodies (11). Results have been mixed from studies investigating the function of phosphorylation using S129A and S129D mutations to respectively block and mimic the modification. Although the phosphorylation mimic was associated with pathology in studies in Drosophila (16) and in transgenic mouse models (17, 18), studies using adeno-associated virus vectors to overexpress α-synuclein in rat substantia nigra found an exacerbation of pathology with the S129A mutation, whereas the S129D mutation was benign, if not protective (19). Interpretation of these studies is complicated by a recent study showing that the S129D and S129A mutations themselves have effects on the aggregation properties of α-synuclein independent of their effects on phosphorylation, with the S129A mutation stimulating fibril formation (20). Clearly, determination of the role of p-Ser-129 phosphorylation would be helped by identification of the responsible kinase. In addition, identification will provide a pathologically relevant way to increase phosphorylation in a cell or animal model.Several kinases have been proposed to phosphorylate α-synuclein, including casein kinases 1 and 2 (10, 12, 21) and members of the G-protein-coupled receptor kinase family (22). In this report, we offer evidence that a member of the polo-like kinase (PLK) family, PLK2 (or serum-inducible kinase, SNK), functions as an α-synuclein kinase. The ability of PLK2 to directly phosphorylate α-synuclein at Ser-129 is established by overexpression in cell culture and by in vitro reaction with the purified kinase. We show that PLK2 phosphorylates α-synuclein in cells, including primary neuronal cultures, using a series of kinase inhibitors as well as inhibition of expression with RNA interference. In addition, inhibitor and knock-out studies in mouse brain support a role for PLK2 as an α-synuclein kinase in vivo.     

Wnt11 Promotes Osteoblast Maturation and Mineralization through R-spondin 2

Wnt11 signals through both canonical (β-catenin) and non-canonical pathways and is up-regulated during osteoblast differentiation and fracture healing. In these studies, we evaluated the role of Wnt11 during osteoblastogenesis. Wnt11 overexpression in MC3T3E1 pre-osteoblasts increases β-catenin accumulation and promotes bone morphogenetic protein (BMP)-induced expression of alkaline phosphatase and mineralization. Wnt11 dramatically increases expression of the osteoblast-associated genes Dmp1 (dentin matrix protein 1), Phex (phosphate-regulating endopeptidase homolog), and Bsp (bone sialoprotein). Wnt11 also increases expression of Rspo2 (R-spondin 2), a secreted factor known to enhance Wnt signaling. Overexpression of Rspo2 is sufficient for increasing Dmp1, Phex, and Bsp expression and promotes bone morphogenetic protein-induced mineralization. Knockdown of Rspo2 abrogates Wnt11-mediated osteoblast maturation. Antagonism of T-cell factor (Tcf)/β-catenin signaling with dominant negative Tcf blocks Wnt11-mediated expression of Dmp1, Phex, and Rspo2 and decreases mineralization. However, dominant negative Tcf fails to block the osteogenic effects of Rspo2 overexpression. These studies show that Wnt11 signals through β-catenin, activating Rspo2 expression, which is then required for Wnt11-mediated osteoblast maturation.Wnt signaling is a key regulator of osteoblast differentiation and maturation. In mesenchymal stem cell lines, canonical Wnt signaling by Wnt10b enhances osteoblast differentiation (1). Canonical Wnt signaling through β-catenin has also been shown to enhance the chondroinductive and osteoinductive properties of BMP2² (2, 3). During BMP2-induced osteoblast differentiation of mesenchymal stem cell lines, cross-talk between BMP and Wnt pathways converges through the interaction of Smad4 with β-catenin (2).Canonical Wnt signaling is also critical for skeletal development and homeostasis. During limb development, expression of Wnt3a in the apical ectodermal ridge of limb buds maintains cells in a highly proliferative and undifferentiated state (4, 5). Disruption of canonical Wnt signaling in Lrp5/Lrp6 compound knock-out mice results in limb- and digit-patterning defects (6). Wnt signaling is also involved in the maintenance of post-natal bone mass. Gain of function in the Wnt co-receptor Lrp5 leads to increased bone mass, whereas loss of Lrp5 function is associated with decreased bone mass and osteoporosis pseudoglioma syndrome (7, 8). Mice with increased Wnt10b expression have increased trabecular bone, whereas Wnt10b-deficient mice have reduced trabecular bone (9). Similarly, mice nullizygous for the Wnt antagonist sFrp1 have increased trabecular bone accrual throughout adulthood (10).Although canonical Wnt signaling regulates osteoblastogenesis and bone formation, the profile of endogenous Wnts that play a role in osteoblast differentiation and maturation is not well described. During development, Wnt11 is expressed in the perichondrium and in the axial skeleton and sternum (11). Wnt11 expression is increased during glucocorticoid-induced osteogenesis (12), indicating a potential role for Wnt11 in osteoblast differentiation. Interestingly, Wnt11 activates both β-catenin-dependent as well as β-catenin-independent signaling pathways (13). Targeted disruption of Wnt11 results in late embryonic/early post-natal death because of cardiac dysfunction (14). Although these mice have no reported skeletal developmental abnormalities, early lethality obfuscates a detailed examination of post-natal skeletal modeling and remodeling.In murine development, Wnt11 expression overlaps with the expression of R-spondin 2 (Rspo2) in the apical ectodermal ridge (11, 15). R-spondins are a novel family of proteins that share structural features, including two conserved cysteinerich furin-like domains and a thrombospondin type I repeat (16). The four R-spondin family members can activate canonical Wnt signaling (15, 17–19). Rspo3 interacts with Frizzled 8 and Lrp6 and enhances Wnt ligand signaling. Rspo1 enhances Wnt signaling by interacting with Lrp6 and inhibiting Dkk-mediated receptor internalization (20). Rspo1 was also shown to potentiate Wnt3a-mediated osteoblast differentiation (21). Rspo2 knock-out mice, which die at birth, have limb patterning defects associated with altered β-catenin signaling (22–24). However, the role of Rspo2 in osteoblast differentiation and maturation remains unclear.Herein we report that Wnt11 overexpression in MC3T3E1 pre-osteoblasts activates β-catenin and augments BMP-induced osteoblast maturation and mineralization. Wnt11 increases the expression of Rspo2. Overexpression of Rspo2 in MC3T3E1 is sufficient for augmenting BMP-induced osteoblast maturation and mineralization. Although antagonism of Tcf/β-catenin signaling blocks the osteogenic effects of Wnt11, Rspo2 rescues this block, and knockdown of Rspo2 shows that it is required for Wnt11-mediated osteoblast maturation and mineralization. These studies identify both Wnt11 and Rspo2 as novel mediators of osteoblast maturation and mineralization.     

Structural and Biochemical Characterization of the Type II Fructose-1,6-bisphosphatase GlpX from Escherichia coli

Gluconeogenesis is an important metabolic pathway, which produces glucose from noncarbohydrate precursors such as organic acids, fatty acids, amino acids, or glycerol. Fructose-1,6-bisphosphatase, a key enzyme of gluconeogenesis, is found in all organisms, and five different classes of these enzymes have been identified. Here we demonstrate that Escherichia coli has two class II fructose-1,6-bisphosphatases, GlpX and YggF, which show different catalytic properties. We present the first crystal structure of a class II fructose-1,6-bisphosphatase (GlpX) determined in a free state and in the complex with a substrate (fructose 1,6-bisphosphate) or inhibitor (phosphate). The crystal structure of the ligand-free GlpX revealed a compact, globular shape with two α/β-sandwich domains. The core fold of GlpX is structurally similar to that of Li⁺-sensitive phosphatases implying that they have a common evolutionary origin and catalytic mechanism. The structure of the GlpX complex with fructose 1,6-bisphosphate revealed that the active site is located between two domains and accommodates several conserved residues coordinating two metal ions and the substrate. The third metal ion is bound to phosphate 6 of the substrate. Inorganic phosphate strongly inhibited activity of both GlpX and YggF, and the crystal structure of the GlpX complex with phosphate demonstrated that the inhibitor molecule binds to the active site. Alanine replacement mutagenesis of GlpX identified 12 conserved residues important for activity and suggested that Thr⁹⁰ is the primary catalytic residue. Our data provide insight into the molecular mechanisms of the substrate specificity and catalysis of GlpX and other class II fructose-1,6-bisphosphatases.Fructose-1,6-bisphosphatase (FBPase,² EC 3.1.3.11), a key enzyme of gluconeogenesis, catalyzes the hydrolysis of fructose 1,6-bisphosphate to form fructose 6-phosphate and orthophosphate. It is the reverse of the reaction catalyzed by phosphofructokinase in glycolysis, and the product, fructose 6-phosphate, is an important precursor in various biosynthetic pathways (1). In all organisms, gluconeogenesis is an important metabolic pathway that allows the cells to synthesize glucose from noncarbohydrate precursors, such as organic acids, amino acids, and glycerol. FBPases are members of the large superfamily of lithium-sensitive phosphatases, which includes three families of inositol phosphatases and FBPases (the phosphoesterase clan CL0171, 3167 sequences, Pfam data base). These enzymes show metal-dependent and lithium-sensitive phosphomonoesterase activity and include inositol polyphosphate 1-phosphatases, inositol monophosphatases (IMPases), 3′-phosphoadenosine 5′-phosphatases (PAPases), and enzymes acting on both inositol 1,4-bisphosphate and PAP (PIPases) (2). They possess a common structural core with the active site lying between α+β and α/β domains (3). Li⁺-sensitive phosphatases are putative targets for lithium therapy in the treatment of manic depressive patients (4), whereas FBPases are targets for the development of drugs for the treatment of noninsulin-dependent diabetes (5, 6). In addition, FBPase is required for virulence in Mycobacterium tuberculosis and Leishmania major and plays an important role in the production of lysine and glutamate by Corynebacterium glutamicum (7, 8).Presently, five different classes of FBPases have been proposed based on their amino acid sequences (FBPases I to V) (9–11). Eukaryotes contain only the FBPase I-type enzyme, but all five types exist in various prokaryotes. Types I, II, and III are primarily in bacteria, type IV in archaea (a bifunctional FBPase/inositol monophosphatase), and type V in thermophilic prokaryotes from both domains (11). Many organisms have more than one FBPase, mostly the combination of types I + II or II + III, but no bacterial genome has a combination of types I and III FBPases (9). The type I FBPase is the most widely distributed among living organisms and is the primary FBPase in Escherichia coli, most bacteria, a few archaea, and all eukaryotes (9, 11–15). The type II FBPases are represented by the E. coli GlpX and FBPase F-I from Synechocystis PCC6803 (9, 16); type III is represented by the Bacillus subtilis FBPase (17); type IV is represented by the dual activity FBPases/inosine monophosphatases FbpA from Pyrococcus furiosus (18), MJ0109 from Methanococcus jannaschii (19), and AF2372 from Archaeoglobus fulgidus (20); and type V is represented by the FBPases TK2164 from Pyrococcus (Thermococcus) kodakaraensis and ST0318 from Sulfolobus tokodai (10, 21).Three-dimensional structures of the type I (from pig kidney, spinach chloroplasts, and E. coli), type IV (MJ0109 and AF2372), and type V (ST0318) FBPases have been solved (10, 11, 19, 20, 22, 23). FBPases I and IV and inositol monophosphatases share a common sugar phosphatase fold organized in five layered interleaved α-helices and β-sheets (α-β-α-β-α) (2, 19, 24). ST0318 (an FBPase V enzyme) is composed of one domain with a completely different four-layer α-β-β-α fold (10). The FBPases from these three classes (I, IV, and V) require divalent cations for activity (Mg²⁺, Mn²⁺, or Zn²⁺), and their structures have revealed the presence of three or four metal ions in the active site.E. coli has five Li⁺-sensitive phosphatases as follows: CysQ (a PAPase), SuhB (an IMPase), Fbp (a FBPase I enzyme), GlpX (a FBPase II), and YggF (an uncharacterized protein) (see the Pfam data base). CysQ is a 3′-phosphoadenosine 5′-phosphatase involved in the cysteine biosynthesis pathway (25, 26), whereas SuhB is an inositol monophosphatase (IMPase) that is also known as a suppressor of temperature-sensitive growth phenotypes in E. coli (27, 28). Fbp is required for growth on gluconeogenic substrates and probably represents the main gluconeogenic FBPase (12). This enzyme has been characterized both biochemically and structurally and shown to be inhibited by low concentrations of AMP (IC₅₀ 15 μm) (11, 29, 30). The E. coli GlpX, a class II enzyme FBPase, has been shown to possess a Mn²⁺-dependent FBPase activity (9). The increased expression of glpX from a multicopy plasmid complemented the Fbp^- phenotype; however, the glpX knock-out strain grew normally on gluconeogenic substrates (succinate or glycerol) (9).In this study, we present the first structure of a class II FBPase, the E. coli GlpX, in a free state and in the complex with FBP + metals or phosphate. We have demonstrated that the fold of GlpX is similar to that of the lithium-sensitive phosphatases. We have identified the GlpX residues important for activity and proposed a catalytic mechanism. We have also showed that YggF is a third FBPase in E. coli, which has distinct catalytic properties and is more sensitive than GlpX to the inhibition by lithium or phosphate.     

Detecting Morphologically Distinct Oligomeric Forms of ��-Synuclein

Neuropathologic and genetics studies as well as transgenic animal models have provided strong evidence linking misfolding and aggregation of α-synuclein to the progression of Parkinson disease (PD) and other related disorders. A growing body of evidence implicates various oligomeric forms of α-synuclein as the toxic species responsible for neurodegeneration and neuronal cell death. Although numerous different oligomeric forms of α-synuclein have been identified in vitro, it is not known which forms are involved in PD or how, when, and where different forms contribute to the progression of PD. Reagents that can interact with specific aggregate forms of α-synuclein would be very useful not only as tools to study how different aggregate forms affect cell function, but also as potential diagnostic and therapeutic agents for PD. Here we show that a single chain antibody fragment (syn-10H scFv) isolated from a phage display antibody library binds to a larger, later stage oligomeric form of α-synuclein than a previously reported oligomeric specific scFv isolated in our laboratory. The scFv described here inhibits aggregation of α-synuclein in vitro, blocks extracellular α-synuclein-induced toxicity in both undifferentiated and differentiated human neuroblastoma cell lines (SH-SY5Y), and specifically recognizes naturally occurring aggregates in PD but not in healthy human brain tissue.Parkinson disease (PD)² is the second most common neurodegenerative disorder of the elderly, affecting more than 500,000 people in the United States (1), with 50,000 new cases reported each year at an annual cost estimated at 10 billion dollars per year. Pathologically, PD is characterized by the progressive loss of dopaminergic neurons in the substantia nigra and formation of fibrillar cytoplasmic inclusions known as Lewy bodies and Lewy neurites (2, 3). The protein α-synuclein has been strongly linked to PD (4, 5) and other related neurodegenerative disorders (6, 7) by several lines of evidence. 1) It is the major component of the hallmark Lewy body aggregates associated with PD. 2) Mutations (A53T, A30P, and E46K, where A30P is human A30P α-synuclein; A53T is human A53T α-synuclein; E46K is human E46K α-synuclein) or multiplication in the α-synuclein gene have been linked to familial PD (8–10). 3) Overexpression of α-synuclein in transgenic mice and Drosophila has been shown to induce the formation of PD-like pathological phenotypes and behavior, although the animal models do not in general replicate neuronal loss patterns (11, 12).α-Synuclein is a small protein (14 kDa) expressed mainly in brain tissues and is primarily localized at the presynaptic terminals of neurons (13). The primary structure of α-synuclein consists of three distinct regions. The N-terminal region of α-synuclein includes the mutation sites associated with familial PD (A53T, A30P, and E46K) and contains six imperfectly conserved repeats (KTKEGV) that may facilitate protein-protein binding. This repeat section is predicted to form amphipathic α-helices, typical of the lipid-binding domain of apolipoproteins (14). The central region, non-amyloid component, is extremely hydrophobic and includes a 12-residue stretch (VTGVTAVAQKTV) that is essential for aggregation (15). The C-terminal region is enriched with acidic glutamate and aspartate residues and is responsible for the chaperone function of α-synuclein (16).α-Synuclein normally exists as an unfolded protein, but it can adopt several different folded conformations depending on the environment, including small aggregates or oligomers, spherical and linear protofibrils, as well as the fibrillar structure found in Lewy bodies (14, 15). A growing body of evidence implicates the oligomeric forms of α-synuclein as the toxic species responsible for neurodegeneration and neuronal cell death (16–18). Several different oligomeric forms of α-synuclein including spherical, annular (19), pore-like (20), and dopamine-stabilized structures have been identified in vitro (21).α-Synuclein is considered a cytosolic protein, and consequently its pathogenic effect was assumed to be limited to the cytoplasm of single cells. However, recent studies have suggested that α-synuclein also has extracellular pathogenic effects (22–25). α-Synuclein was detected in blood plasma and cerebrospinal fluid in both monomeric and oligomeric forms (22–25), and the presence of significantly elevated levels of oligomeric species of α-synuclein has been reported extracellularly in plasma and cerebrospinal fluid samples from patients with PD (23). Furthermore, various studies have shown that aggregated α-synuclein added extracellularly to the culture medium is cytotoxic (26–32).Despite all these studies, it is still not clear how the various aggregate forms of α-synuclein are involved in the progression of PD. Therefore, reagents that can interact with specific aggregate forms of α-synuclein would be very useful not only for fundamental studies of how α-synuclein aggregates affect cell function but also as potential diagnostic and therapeutic agents for PD.Recently, we reported inhibition of both aggregation and extracellular toxicity of α-synuclein in vitro by a single chain variable domain antibody fragment (scFv) that specifically recognized an oligomeric form of α-synuclein (32). In this study, we describe a second scFv (syn-10H) that binds a larger later stage oligomeric form of α-synuclein than the previously reported scFv. The syn-10H scFv neutralizes α-synuclein-induced toxicity in both undifferentiated and differentiated SH-SY5Y human neuroblastoma cell line and inhibits α-synuclein aggregation in vitro. The syn-10H scFv reacts specifically with homogenized PD brain tissue but does not cross-react with similarly treated samples taken from Alzheimer disease (AD) or healthy brain samples. Such scFvs therefore have potential value as diagnostic reagents to identify the presence of specific oligomeric species in PD tissue and fluid samples. The scFvs also have value as therapeutic agents as they can be used either extracellularly or expressed intracellularly (intrabodies) to prevent formation of toxic aggregates in vivo whether inside or outside of cells. Intrabodies have been used efficiently to neutralize toxic effects of different pathogenic agents, including α-synuclein (33–36). Moreover, immunization studies in mouse models of PD have shown that extracellular antibodies can reduce accumulation of intracellular aggregates of α-synuclein (37), thereby providing precedent for the use of scFvs in potential passive vaccination strategies for treating PD.