Translational control by cytoplasmic polyadenylation in Xenopus oocytes

Elongation of the poly(A) tails of specific mRNAs in the cytoplasm is a crucial regulatory step in oogenesis and early development of many animal species. The best studied example is the regulation of translation by cytoplasmic polyadenylation elements (CPEs) in the 3′ untranslated region of mRNAs involved in Xenopus oocyte maturation. In this review we discuss the mechanism of translational control by the CPE binding protein (CPEB) in Xenopus oocytes as follows:1.The cytoplasmic polyadenylation machinery such as CPEB, the subunits of cleavage and polyadenylation specificity factor (CPSF), symplekin, Gld-2 and poly(A) polymerase (PAP).2.The signal transduction that leads to the activation of CPE-mediated polyadenylation during oocyte maturation, including the potential roles of kinases such as MAPK, Aurora A, CamKII, cdk1/Ringo and cdk1/cyclin B.3.The role of deadenylation and translational repression, including the potential involvement of PARN, CCR4/NOT, maskin, pumilio, Xp54 (Ddx6, Rck), other P-body components and isoforms of the cap binding initiation factor eIF4E.
1
Introduction
The regulation of translation of mRNAs by cytoplasmic elongation of the poly(A) tail was discovered some twenty years ago in the oocytes and early embryos of clams, worms, frogs and mice [1–6]. These maternal mRNAs are stored in the growing oocyte with a short poly(A) tail of 20 to 40 nucleotides and are translationally repressed (masked). Upon oocyte maturation or after fertilisation, the poly(A) tail of masked mRNAs is elongated to 80–250 residues and the mRNAs are translationally activated. A long poly(A) tail is thought to be stimulatory to translation through the binding of cytoplasmic poly(A) binding proteins, which recruit initiation factors and form a closed-loop complex through their association with the translation initiation factor eIF-4G [7]. Cytoplasmic polyadenylation is essential for the meiotic maturation of the oocyte in both Xenopus and mouse, as it mediates the translational activation of the mRNAs encoding c-Mos kinase and mitotic cyclins [8]. In addition, cytoplasmic polyadenylation has been implicated in the cell cycle, in cellular senescence and in the formation of memory through synaptic plasticity [9]. In this review we will discuss the current knowledge of the mechanism of translational control by cytoplasmic polyadenylation, with an emphasis on Xenopus oocyte maturation and CPEB, the cytoplasmic polyadenylation element binding protein. A review of the role of cytoplasmic polyadenylation in mammalian gametogenesis is provided elsewhere in this volume (Tadashi Baba).
2
mRNA elements and RNA binding proteins
In order for mRNAs to be singled out for cytoplasmic polyadenylation, they have to be recognised by the polyadenylation machinery. In oocytes and early embryos of the African clawed frog, Xenopus, cytoplasmic polyadenylation can be conferred by several discrete elements in the 3′ untranslated region (3′ UTR) of the mRNA which are recognised by specific RNA binding proteins. At least 4 different cytoplasmic polyadenylation elements have been identified in Xenopus. The C-rich cytoplasmic polyadenylation element and the U-rich embryonic cytoplasmic polyadenylation element normally mediate cytoplasmic polyadenylation in the zygote and early embryo [10,11]. The putative cytoplasmic polyadenylation factors binding to these elements are poly(rC) binding protein 2 (PCBP2) and Elav related protein A, the ortholog of HuR, respectively [10,12–14]. A polyadenylation element that functions early in oocyte maturation, the polyadenylation response element (PRE), was predicted computationally [15,16]. The RNA binding protein Musashi was shown to bind to some, but not all PREs and mutating Musashi or its known consensus binding site blocked cytoplasmic polyadenylation, indicating that it is likely to be a cytoplasmic polyadenylation factor [17]. These data suggest that the collection of cytoplasmic polyadenylation elements that have been called PREs may represent a mixture of Musashi binding sites and at least one more uncharacterised cytoplasmic polyadenylation element.
By far the best characterised cytoplasmic polyadenylation element is the CPE, which is required for the cytoplasmic polyadenylation of a number of mRNAs, including cyclin B1 mRNA, during oocyte maturation and during the embryonic cell cycle [18–20]. The consensus CPE is often described as U5AU, but variations such as U4AU (c-mos), and U4–5A2U (cyclin B1) have also been shown to be active [5,6,21,22]. The CPEs in the mRNA encoding mouse c-Mos are active in mature Xenopus oocytes, but only conform to U5A [23,24]. Strongly polyadenylating mRNAs, however, generally adhere to the U5A1–2U consensus. In addition, examination of the cases in the existing literature suggests that CPEs that mediate significant polyadenylation tend to be close to the polyadenylation signal, from downstream to overlapping and up to approximately 60 nt upstream [21,25–29].
The CPE binds cytoplasmic polyadenylation element binding protein (CPEB), an RNA binding protein containing two RNA recognition motifs and a zinc finger region, all of which are required for recognition of the CPE [21,30,31]. In addition to the fact that many mRNAs bearing a CPE undergo cytoplasmic polyadenylation, a wealth of evidence indicates that CPEB is required for cytoplasmic polyadenylation. Depletion of CPEB from an egg extract abolishes polyadenylation and injection of a CPEB antibody blocks it in oocytes and embryos [20,21,31,32]. N-terminal deletions and phosphorylation site mutants of CPEB act as dominant negative blockers of cytoplasmic polyadenylation [20,21,31,32]. In mice, knock out or knock down of Cpeb1 causes poly(A) tail changes in target mRNAs in early oogenesis and oocyte maturation [33,34]. CPEB is therefore the only well-established mRNA specificity factor for cytoplasmic polyadenylation in vertebrates.
In addition to an mRNA specific element, the nuclear poly(A) signal (AAUAAA or AUUAAA) is absolutely required for cytoplasmic polyadenylation [5,6,11,22,28]. The poly(A) signal is thought to recruit a variant of the cleavage and polyadenylation specificity factor (CPSF), a four subunit complex that binds this element and mediates polyadenylation in the nucleus [35,36]. The 160 kDa subunit is the RNA binding protein that recognises the poly(A) signal [37], while the 73 kDa subunit is the endonuclease that mediates the formation of the 3′ ends of all mRNAs [38–41]. The 100 kDa subunit resembles the 73 kDa subunit but does not appear to have nuclease activity and its precise function in 3′ end processing is unknown. The 30 kDa subunit is a zinc finger protein that has been reported to have some affinity for RNA and it potentially has endonuclease activity. It was recently shown to mediate interactions between CPSF and the body of RNA polymerase II [42–45]. An additional CPSF associated factor, Fip1, has been shown to mediate the interaction with poly(A) polymerase and have U-rich RNA binding activity [46]. It has probably been present in most of the previously utilised preparations of CPSF and is thought of as a fifth subunit by many in the field [40,46].
The 100 and 30 kDa CPSF subunits are present in the Xenopus oocyte cytoplasm, and the 160 kDa subunit has been inferred to be present from its binding activity, while the 73 kDa endonuclease is absent from the cytoplasmic CPSF complex [47]. It is unknown whether Fip1 is present in the oocyte cytoplasm. Immunodepletion of the 100 kDa subunit from egg extracts or expression of a viral protein that blocks nuclear polyadenylation by binding to the 30 kDa subunit also abolishes cytoplasmic polyadenylation, demonstrating that CPSF is required for this process [47]. In addition, CPEB is coimmunoprecipitated with the 100 kDa CPSF subunit from oocyte extracts (but not with the 73 kDa subunit) and it binds to the 160 kDa subunit in reticulocyte lysate [47,48]. Whether these interactions are direct or mediated by other proteins present in both oocyte extract and reticulocyte lysate is not yet clear. Curiously, the binding of CPSF to the poly(A) signal is stimulated by a CPE even in the absence of CPEB, a phenomenon that could be mediated by the RNA binding activity of the 30 kDa subunit or by Fip1 [46,49]. However, CPEB strongly stimulates polyadenylation of a CPE containing RNA in a pure reaction system containing nuclear CPSF and poly(A) polymerase [48]. It is therefore likely that CPEB improves the recruitment of CPSF to the mRNA by binding directly to both.
3
Symplekin
Symplekin is a protein found in nuclear complexes containing CPSF and other processing factors and is thought of as a scaffold protein involved in 3′ end RNA processing [50–52]. In yeast and plants, homologues of symplekin are required for proper cleavage and polyadenylation, and this is possibly also the case in vertebrates [53–55]. In vertebrate somatic cells symplekin is predominantly nuclear and in epithelial cells also localised at tight junctions [50,56]. In the Xenopus oocyte, however, symplekin is also found in the cytoplasm in complexes with the 100 kDa subunit of CPSF [50]. A symplekin antibody also precipitates CPEB from oocyte extracts, indicating that the protein is in cytoplasmic polyadenylation complexes [57]. Moreover, the symplekin antibody inhibits cytoplasmic polyadenylation in oocytes and extracts [57]. These data strongly suggest that symplekin is involved in cytoplasmic polyadenylation.
4
The cytoplasmic poly(A) polymerase
Initially, a form of one of the classical nuclear poly(A) polymerases was thought to be responsible for cytoplasmic polyadenylation too. In vitro reconstitution experiments indicated that the poly(A) polymerase (PAP) isolated from bovine thymus can mediate CPE and poly(A) signal dependent polyadenylation [47–49]. Cytoplasmic poly(A) polymerase species were detected in Xenopus oocytes and an isoform was cloned that was 87% identical to bovine PAP but lacked the nuclear localisation signal [58,59]. Three different polyclonal antibodies against this protein inhibited polyadenylation in egg extracts [58]. However, after these findings the work on the cytoplasmic poly(A) polymerase stagnated. No native complex containing CPEB and a PAP isoform was ever reported and studies on the regulation of PAP activity indicated that the enzyme is inactivated by phosphorylation during oocyte maturation, when cytoplasmic polyadenylation is actually induced [60].
In 2002, a Caenorhabditis elegans germline determinant called gld-2 was cloned and shown to encode a cytoplasmic poly(A) polymerase that is recruited to certain mRNAs by an RNA binding protein [61]. The Gld-2 protein belongs to the same large family of DNA polymerase β nucleotidyl transferases, but only has limited additional homology to the classical poly(A) polymerases and it lacks the RNA binding domain. It soon transpired that this protein is widely conserved, having homologues in fission yeast and mammalians [62,63].
The Xenopus Gld-2 protein (xGld-2) coimmunoprecipitated very efficiently with symplekin in both mature and immature oocytes. In addition, tagged xGld-2 was shown to bind the 160 kDa CPSF subunit as well as CPEB synthesised in reticulocyte lysate [57]. This indicates that xGld-2 can form complexes with symplekin, CPSF and CPEB, although the direct interactions are as yet unclear. Gld-2 overexpressing oocytes and extracts supplemented with Gld-2 displayed an increase in their CPE-dependent polyadenylation and symplekin depleted extracts regained some polyadenylation activity if they were replenished with recombinant CPEB, symplekin and Gld-2 [57].
The less than optimal reconstitution of cytoplasmic polyadenylation by Gld-2 may indicate that cytoplasmic PAP or an unknown additional cytoplasmic polyadenylation factor is being depleted with symplekin. However, it is also very plausible that not all the supplemented proteins are completely active. Especially CPEB is notoriously difficult to obtain in a soluble form and isolation procedures usually include a denaturing step [30]. On the whole, the evidence seems to indicate that xGld-2 is an authentic cytoplasmic poly(A) polymerase in Xenopus oocytes, but involvement of the cytoplasmic PAP can as yet not be excluded. In Drosophila, PAP has been implicated in cytoplasmic polyadenylation in embryos [64], and although initial experiments implicated mouse Gld-2 in cytoplasmic polyadenylation in oocytes, polyadenylation appeared unimpaired in a knock out mouse [65,66]. The cytoplasmic polyadenylation mechanisms in mammalian germ cells are discussed in more detail by T. Baba elsewhere in this volume.
What part of the cytoplasmic polyadenylation complex recruits either PAP or Gld-2 is also still an open question. It is possible that CPEB replaces Fip1 in cytoplasmic polyadenylation, or that another specific recruitment factor exists for Gld-2. Also, the nuclear cleavage stimulation factor CstF77 has been reported to be present in the cytoplasmic polyadenylation complex in Xenopus oocytes, but appears to have an as yet undefined role in translational repression, rather than in cytoplasmic polyadenylation [67].
In nuclear polyadenylation, binding of the nuclear poly(A) binding protein, PABP2, is required to limit the length of the poly(A) tail. It has been suggested that such a function is required in cytoplasmic polyadenylation, as symplekin immunoprecipitates from polyadenylating egg extracts add abnormally long poly(A) tails [57]. It is unknown if the length of the poly(A) tail added is controlled in cytoplasmic polyadenylation, but it is worth noting in this context that the main poly(A) binding protein in oocytes is not the somatic cytoplasmic PABP, but an embryo specific protein, ePABP, which may have a specific function in this context [68,69].
Recently, it was shown that ePAB and somatic PABP both can bind to CPEB, and that this interaction is reduced by phosphorylation of CPEB during oocyte maturation [65,70]. The ePABP–CPEB interaction has been proposed to stimulate cytoplasmic polyadenylation and translational activation, as demonstrated by depletion experiments [65]. ePABP may therefore be a cytoplasmic polyadenylation factor. However, the data presented appear to show that symplekin is partially depleted with ePABP, which could explain the reduction in polyadenylation. The add-back experiment does unfortunately only show a minimal restoration of polyadenylation. Depletion of CPEB with ePABP is described for experiments described in the discussion section of this paper, adding to these concerns. In addition, because most panels in this paper do not show direct comparisons between CPE containing and CPE lacking RNA substrates, the intriguing effects of ePABP depletion could perhaps also be explained by a general effect of enhanced deadenylation and reduced translation of all polyadenylated mRNAs, which are predicted effects of low ePABP levels [68,69]. For the moment, it therefore appears prudent to reserve judgement on the function of the CPEB–ePABP interaction.
5
The activation of cytoplasmic polyadenylation
In Xenopus oocytes, meiotic maturation is induced by steroid hormones, probably through the action of both classical nuclear steroid receptors and a G protein coupled transmembrane receptor [71–73]. A rapid drop in cyclic AMP leads to the inactivation of protein kinase A, which is necessary for the induction of cytoplasmic polyadenylation. In recent years, progress has been made in various aspects of the activation mechanisms of cytoplasmic polyadenylation, although the picture is far from complete.
Tethering of mammalian Gld-2 to an mRNA using a fusion with an RNA binding domain and an mRNA with a binding site causes strong polyadenylation in immature Xenopus oocytes, indicating that the protein is constitutively active and only needs to be recruited to the mRNA to elongate the poly(A) tail [62]. However, as described above, Gld-2 is already associated with the polyadenylation factors in immature oocytes [57]. In addition, Gld-2 overexpression also leads to robust polyadenylation of CPE containing RNA in immature oocytes, which implies that the protein is probably inactivated by factors that are titrated by the overexpressed protein [57]. Since the action of Gld-2 requires recruitment to the RNA for activity, the titrated factor is unlikely to be CPEB itself. An intriguing possibility is that the protein depleted by Gld-2 is the cytoplasmic isoform of poly(A) ribonuclease (PARN). A 64 kDa catalytically active fragment of PARN has been found to coimmunoprecipitate with symplekin, CPEB and Gld-2 in immature but not in mature oocytes and binds to CPEB and Gld-2 synthesised in reticulocyte lysate [74]. The full length PARN is not abundantly found in this complex. The 64 kDa PARN isoform is thought to be responsible for the short poly(A) tails of CPE containing mRNAs, as a PARN antibody inhibited CPE-mediated deadenylation in immature oocytes [74]. In addition, catalytically inactive PARN can induce polyadenylation of CPE containing RNAs in immature oocytes and overexpression of wild type PARN represses the polyadenylation induced by Gld-2 overexpression [74]. These data indicate that PARN is the repressive factor depleted by Gld-2 injection and that it is the ejection of PARN from the polyadenylation complex that induces cytoplasmic polyadenylation [74]. There are two possibilities to explain the action of PARN on CPE containing mRNAs, either Gld-2 continuously adenylates while PARN takes these nucleotides off, or the presence of PARN blocks Gld-2 activity, for instance by preventing access to the 3′ end of the mRNA.
Since not all mRNAs are activated by cytoplasmic polyadenylation at the same time, the activation of the complexes must depend at least in part on mRNA specificity factors. In maturing Xenopus oocytes, cytoplasmic polyadenylation has been classed into 2 types, early (class I) and late (class II). Early polyadenylating mRNAs gain a poly(A) tail well before the activation of cdk1/cyclin B and the breakdown of the nuclear envelope (germinal vesicle breakdown, GVBD). Early polyadenylation is independent of the synthesis of the kinase c-Mos (which is itself encoded by a class I mRNA), while late polyadenylating mRNAs are dependent on c-Mos synthesis and the subsequent cdk1/cyclin B activation and gain their tails after GVBD [16,75,76]. The less well characterised polyadenylation response element (PRE, see above) and its binding factor Musashi have been shown to be responsible for some of the early polyadenylation events, despite the fact that these mRNAs also contain CPEs [15,17]. However, there is a clear correlation between the placement of the CPEs in an mRNA and the timing of its polyadenylation [15,77,78]. In addition, some late polyadenylating mRNAs also contain PREs/Musashi binding sites and the histone B4 mRNA, which contains both a PRE and a CPE, has been reported to be polyadenylated early, but has been shown to be Mos-dependent, indicating that the current classification in early and late perhaps oversimplifies the situation [15,17,75,78].
The evidence for which factor mediates early polyadenylation rests primarily on the use of dominant negative Musashi and CPEB mutants, which block translation and polyadenylation of c-Mos mRNA and GVBD [15,17,48]. Charlesworth et al. compared Musashi and CPEB mutants, and came to the conclusion that Musashi alone mediates the early polyadenylation, in contrast to earlier findings by Mendez et al., which implicated CPEB in the early polyadenylation [17,32]. The CPEB phosphorylation mutant used by Charlesworth et al. is a rather weak dominant negative (L.E. Hake, Boston, USA, unpublished observation), so it is possible that CPEB-dependent early polyadenylation is not completely repressed by it in all cases. Differences in expression levels and examination of endogenous or injected RNAs could also explain the discrepancies between the two studies. In addition, either or both dominant negative proteins could be titrating other cytoplasmic polyadenylation factors and inhibit polyadenylation on mRNAs to which they do not normally control, or they could fail to enter a specific mRNA–protein complex due to low exchange rates. Importantly, injection of a CPEB antibody prevented GVBD and gave strong repression of polyadenylation of injected early RNAs, including a Mos 3′ UTR construct, indicating that CPEB-mediated early polyadenylation exists [21]. In addition, interference with pathways that are known to affect CPEB phosphorylation and activation leads to a reduction in pre-GVBD c-Mos mRNA polyadenylation and a delay or block in oocyte maturation, including in those papers that claim that early polyadenylation is CPEB-independent [15,16,32,79,80]. However, there remains the possibility that the same signal transduction pathways ultimately activate Musashi and CPEB, which would make it impossible to distinguish between the effects of the two proteins in these experiments, with the possible exception of the CPEB antibody injection. With the limitations of the currently available evidence, it seems safest to assume that Musashi and CPEB both contribute to the polyadenylation of early mRNAs and that some mRNAs may be exclusively dependent on one or the other. Further work is required to resolve this issue definitively.
The induction of Xenopus oocyte maturation and early cytoplasmic polyadenylation by progesterone is dependent on a drop in cAMP levels and the resulting inhibition of protein kinase A [17,81]. Downstream of this event is the phosphorylation of CPEB on Ser174, which happens early in oogenesis. Although CPEB, CPSF and Gld-2 are already in a complex in immature oocytes, this phosphorylation appears to induce a stronger association of CPEB with CPSF and Gld-2 [32,48,82]. Importantly, Ser174 phosphorylation also induces the ejection of PARN from the polyadenylation complex [74]. Mutation of Ser174 to alanine results in a dominant negative protein that inhibits oocyte maturation and early polyadenylation partially or fully and late polyadenylation completely [15,32,83]. In addition, injection of a peptide containing the phosphorylation site delays GVBD considerably, suggesting that it is inhibiting an early polyadenylation event [32]. All evidence therefore indicates that phosphorylation of Ser174 is a crucial event in the activation of cytoplasmic polyadenylation.
There is some controversy surrounding the kinase mediating the early phosphorylation of CPEB on Ser174. Initially, it was demonstrated that Aurora A kinase (Eg2) can phosphorylate this site and that this requires the N-terminus of CPEB, in addition to the phosphorylation site itself [32]. This suggested that the N-terminally deleted CPEB may owe its dominant negative properties to the lack of a binding site for Aurora A. In addition, in vitro polyadenylation reactions containing recombinant CPEB, purified CPSF and nuclear PAP are strongly stimulated by the addition of Aurora A [48]. Glycogen synthase kinase 3 (GSK3) is thought to negatively regulate Aurora A by phosphorylation and this block appears to be relieved during oocyte maturation [83]. Consistent with a role in early oocyte maturation, GSK3 overexpression inhibited GVBD in Xenopus oocytes [83]. However, studies from three independent groups have failed to detect Aurora A activation early in oocyte maturation [79,84–86]. In addition, Aurora A inhibitors do not affect oocyte maturation or CPEB phosphorylation and depletion of Aurora A from oocyte extracts does not inhibit early CPEB phosphorylation either [79]. There is therefore sufficient evidence to throw serious doubt on the role of Aurora A in the activation of CPE-mediated cytoplasmic polyadenylation early in oogenesis. In mouse neurons, calmodulin dependent kinase II (CamKII) can phosphorylate the corresponding site on mouse CPEB, Thr171 [87]. CamKII plays a role in the induction of mouse oocyte maturation and is known to be present in Xenopus eggs, and could therefore be an alternative candidate kinase for mediating the early phosphorylation [88–91]. Because CamKII can be activated independently of calcium, the absence of a calcium flux in early oocyte maturation is not necessarily an obstacle for this hypothesis [92]. Evidence for a requirement of CamKII activity early in oocyte maturation would be necessary to make this more than speculation. However, Aurora A is very active later in oogenesis and it is undeniably capable of activating CPEB. It therefore appears likely that at the very least the maintenance of CPEB phosphorylation on Ser174 is carried out by this kinase.
Mitogen activated protein kinase (MAP kinase) has been implicated in the activation of early CPE-mediated, but not PRE-mediated, cytoplasmic polyadenylation [16,17,79,93]. An early, c-Mos independent activation of MAP kinase has been detected during oocyte maturation and CPEB is phosphorylated by MAP kinase on multiple sites [32,79,94]. MAP kinase does not phosphorylate Ser174, but has been suggested to either prime CPEB for Ser174 phosphorylation or to activate the Ser174 kinase [79]. As the early MAPK activation is dependent on protein synthesis, translational activation of another mRNA is probably required [94]. One potential candidate is RINGO/Speedy, a Cdk1 interacting and activating protein that is transiently expressed after progesterone treatment and required for oocyte maturation and CPEB phosphorylation [65,95–98].
A guanine nucleotide exchange factor for the Rho family of GTPases was found to interact with the N-terminal half of CPEB in a yeast 2 hybrid screen and called XGef [80]. This interaction was confirmed in oocytes and has been shown to be direct [80,99]. There appear to be 2 binding sites for CPEB on XGef [99]. Antibodies against XGef block oocyte maturation and prevent the polyadenylation and translation of c-Mos mRNA [80,99]. Conversely, overexpression of XGef accelerates oocyte maturation and c-Mos polyadenylation, independent of the production of c-Mos protein, indicating that it is upstream of this kinase in the signal cascade of meiotic maturation [80]. XGef enhances the early phosphorylation of CPEB and the DH domain associated with nucleotide exchange activity is required for its effects of on cytoplasmic polyadenylation [80,99]. In addition, a mutant of XGef that retains nucleotide exchange activity but has impaired binding to CPEB reduced early CPEB phosphorylation and delayed oocyte maturation [99]. However, a broad spectrum Rho GTPase inhibitor did not affect oocyte maturation or CPEB phosphorylation, indicating that the DH domain may not function to activate a Rho family GTPase, but is required in another capacity [79]. Strikingly, XGef immunoprecipitates were found to contain MAPK in both immature and mature oocytes and it therefore may be required to bring CPEB to the signalling complexes involved in its phosphorylation [79].
In another yeast 2 hybrid study, mouse CPEB was found to bind the small intracellular domain (ICD) of the transmembrane protein amyloid precursor like protein 1 (APLP1) and its relatives [100]. In Xenopus oocytes, Gld-2, the CPSF 100 kDa subunit and symplekin were predominantly associated with the plasma membrane in the same fractions as overexpressed APLP1 and immunoprecipitation of CPEB from these fractions indicates that it can associate with APLP1 [100]. Immunogold electron microscopy confirmed that CPEB and CPE containing RNA is associated with membranes. Overexpression of full length APLP1 induced some cytoplasmic polyadenylation in untreated oocytes and enhanced the effect of low concentrations of progesterone. The ICD alone had even stronger effects, indicating that the action of APLP1 is not dependent on membrane localisation. After treatment with low concentrations of progesterone, APLP1 stimulated CPEB phosphorylation on Ser174 [100]. While the association with amyloid precursor proteins may have great significance for the role of CPEB and cytoplasmic polyadenylation in neurons, it is as yet unclear whether APLP1 or its relatives are required for cytoplasmic polyadenylation in oocytes and whether it mediates the membrane association of the cytoplasmic polyadenylation complexes.
Late cytoplasmic polyadenylation is required for progression from meiosis I to meiosis II during oocyte maturation [101]. mRNAs that undergo late polyadenylation often have a CPE overlapping with their poly(A) signal, e.g. UUUUAAUAAA [15,77,78]. The late polyadenylation during oocyte maturation is dependent on the activation of the mitotic kinase cdk1 and its regulatory cyclin subunits [16,77,78,93]. Cdk1 does phosphorylate CPEB leading to degradation of most of the oocyte CPEB via ubiquitin-mediated degradation [101–103]. This degradation is required for late polyadenylation to occur. Remarkably, symplekin bound CPEB appears to represent the stable fraction of CPEB, as symplekin immunoprecipitates do not show a difference in CPEB content between immature and mature oocytes [57]. This implicates that CPEB in polyadenylation complexes is stable during oocyte maturation, consistent with the polyadenylation activity observed. A model for late cytoplasmic polyadenylation can be devised in which the abundant ‘free’ CPEB (i.e. CPEB in a different complex than symplekin, CPSF and the poly(A) polymerase) binds to the CPE overlapping the poly(A) signal in late polyadenylating mRNAs and prevents the recruitment of CPSF and symplekin by the polyadenylation signal early in oocyte maturation. After cdk1 activation, most free CPEB is depleted and a complex containing CPEB, symplekin and CPSF binds both the CPE and the poly(A) signal [101].
A current view of CPE-mediated cytoplasmic polyadenylation in oocyte maturation is depicted in Figs. 1 and 2 and can be summarised as follows:1.The activation of CPE-mediated cytoplasmic polyadenylation during progesterone induced oocyte maturation is induced by a drop in protein kinase A activity and requires an early translation event, perhaps translation of RINGO/Speedy.2.This induces the early activation of MAP kinase, which is associated with the polyadenylation complex through XGef and phosphorylates CPEB on multiple sites, but not on Ser174.3.CPEB is phosphorylated on Ser174 by an as yet not fully confirmed kinase, possibly Aurora A or CamKII, which is required for the induction of CPE-mediated polyadenylation and causes an increase in the binding between Gld-2, CPSF and CPEB, causing the ejection of PARN from the complex and allowing Gld-2 to elongate the poly(A) tail of the mRNA.4.After GVBD, CPEB is phosphorylated by cdk1 and the free CPEB is mostly degraded, allowing the CPEB in polyadenylation complexes (now probably phosphorylated on Ser174 by Aurora A) to activate cytoplasmic polyadenylation on mRNAs which contain a CPE overlapping with the poly(A) signal.
A surprising number of proteins involved in the activation of polyadenylation are themselves upregulated by this process. This should create positive feedback loops which amplify the signal and contribute to the progression of meiotic maturation. As discussed above, the mRNA for the serine/threonine kinase c-Mos is one of the early targets of cytoplasmic polyadenylation [16,75,76]. It is an activator of the MAPK kinase MEK, and its synthesis leads to further activation of MAP kinase, activation of cdk1 and phosphorylation of CPEB [71]. The mitotic cyclins, activators of cdk1, are induced by cytoplasmic polyadenylation, as is Aurora A [19,26]. Similarly, CamKII is regulated by cytoplasmic polyadenylation in neurons [104]. Finally, Gld-2 targets its own mRNA, which contains CPEs in its 3′ UTR[105]. A system with so much positive feedback will require strong brakes, and it appears that at least in some cases this is provided by deadenylation factors, as discussed below.
6
Deadenylation and translational repression
The maternal mRNAs that are stored in the oocyte in an untranslated state have short poly(A) tails, but have been reported to have normal poly(A) tail addition in the nucleus in both mouse and frog [74,106]. In both organisms, the deadenylation of RNA injected into the cytoplasm requires CPEs, indicating that the CPEB–PARN complex, discussed above, mediates this process [74,106]. However, RNA substrates containing CPEs alone have not been tested in these experiments and it is therefore not clear if a CPE is sufficient for deadenylation or if other sequence elements are required. In fact almost all CPE containing mRNAs have putative binding sites for other deadenylation factors.
Firstly, the deadenylation factor Pumilio binds directly to CPEB, and therefore could contribute to the deadenylation of all CPE containing mRNAs [107,108]. Many CPE containing mRNAs also contain Pumilio binding sites, including cyclin B1 and Gld-2 [97,107–109], indicating that the recruitment of Pumilio maybe both by protein–protein and RNA–protein associations. Pumilio is a member of a highly conserved family of RNA binding proteins called the Puf family. Members of this family can mediate translational repression and mRNA destabilisation in organisms from yeast to vertebrates [110,111]. In yeast, the Puf family protein Mpt5 interacts with the conserved deadenylase complex CCR4–Pop2–Not, by binding to Pop2/Caf1 [112–114]. This association was also confirmed for the human and worm proteins [113]. In addition, Pumilio is known to aid the recruitment of the translational repressor Nanos, both in fly and frog [107,115]. Nanos can recruit the CCR4–Pop2–Not complex by binding to Not4 and contributes to the translational repression of fly cyclin B [116]. So far, the role of the CCR4–Pop2–Not complex in poly(A) tail metabolism has not been studied in Xenopus oocytes and embryos, but the conservation of both the recruitment proteins and one of the target mRNAs (cyclin B) seems to indicate that it is very likely to play a role.
A subset of mRNAs that are activated by CPE-mediated cytoplasmic polyadenylation during oocyte maturation loose their poly(A) tails after fertilisation [19,117,118]. In most cases, this is mediated by an embryonic deadenylation element (EDEN) in the 3′ UTR of the mRNA, which binds the deadenylation factor EDEN-BP [119]. Aurora A, cyclin B1 and c-Mos encoding mRNAs all bind EDEN-BP and the 3′ UTR of Gld-2 mRNA contains good consensus binding sites, making EDEN-BP a good candidate repressor of the cytoplasmic polyadenylation-mediated positive feedback loops during early embryogenesis [105,119–121]. The mammalian ortholog of EDEN-BP, CUGBP1, can recruit PARN [122]. A similar association of EDEN-BP and PARN in the Xenopus embryo could explain its role in deadenylation, but so far this complex has not been reported. It can therefore not be excluded that EDEN-BP mediates its deadenylation activity in Xenopus embryos through another deadenylase, for instance the CCR4–Pop2–Not complex. No direct contacts between CPEB containing and EDEN containing complexes has been described so far. Because CPEs, EDEN sequences, Musashi and Pumilio binding sites often occur in the same 3′ UTR, their associated complexes can be expected to compete for the end of the mRNA to mediate deadenylation or polyadenylation.
Although a short poly(A) tail will lead to inefficient translation of an mRNA, it is not sufficient for complete translational repression, as illustrated by the partial polysomal association of histone B4 mRNA and the accumulation of B4 protein in Xenopus oocytes [78,123,124]. Many of the other stored maternal mRNAs have to be strongly translationally repressed to enable the subsequent stages of growth, maturation and fertilisation of the oocyte [71]. CPEs, Musashi and Pumilio binding sites can repress translation of specific mRNAs, often in the absence of a poly(A) tail or in the absence of deadenylation, indicating that they have an additional function in blocking translation [75,105,107–109,125–127]. Several detailed models exist to explain the translational repression by CPEs, some of which are presented in Fig. 3.
In full grown oocytes, a CPEB binding factor called maskin was found to associate with the cap binding initiation factor eIF4E in a manner that should preclude recruitment of eIF4G and thus inhibit translation [128]. Maskin is a homologue of the transforming acidic coiled-coil domain protein 3 (TACC3) and these proteins play a vital role in the formation of the mitotic spindle in multiple organisms [129–134]. The binding between maskin and eIF4E is weak but detectable and this association is abrogated by binding of PABP to the poly(A) tail elongation and by phosphorylation of maskin in both oocytes and embryos [129,135–137]. As the eIF4E binding site in maskin appears not to be present in other organisms, it is not clear how widespread this regulation is [138]. A more conserved CPEB associated eIF4E binding protein is the neuronal protein neuroguidin, which has been proposed to function in a similar manner to maskin in repressing translation in the nervous system [139].
The deadenylase PARN is a well characterised cap binding protein and a 5′ cap structure is required for deadenylation by PARN [140–142]. CPEB can recruit PARN to the mRNA, but deadenylation still requires a cap structure [74]. This leads to a conflict between the maskin model and the opposing polymerase-deadenylase model [74,128]. Either the maskin model is true and eIF4E/maskin is stably bound to the cap and PARN is not continuously active, or the opposing polymerase-deadenylase model is correct and CPEB bound PARN is stably bound to the cap and continuously active, excluding eIF4E from the cap. In fact, PARN could very well mediate both the deadenylation and the translational repression of mRNAs to which it is recruited by excluding eIF4E from the mRNA [120].
Both maskin and PARN are absent in early oocytes, so other repression mechanisms must exist to mediate translational repression in these cells. Recently, a large CPEB containing RNP complex was found in early oocytes [143]. This complex contains several proteins found in P-bodies and related RNP granules that have been implicated in both translational control and mRNA degradation [144]. In mammalian cells, CPEB can also be found in large RNP granules including P-bodies [145]. The early oocyte CPEB complex contains the eIF4E variant eIF4E1b, the eIF4E binding protein 4E-T, the RNA helicase Xp54 (Ddx6, Rck, Me31B, Dhh1) and the P-body components p100 (Pat1) and Rap55 (Scd6, CAR-1). It does not contain PARN, maskin or the canonical cap binding initiation factor eIF4E1a. Rap55 and Xp54 can mediate translational repression in Xenopus oocytes when tethered to an mRNA [146–148]. In addition, the yeast homologues of both proteins, as well as of p100, are involved in general repression of translation [149]. Strikingly, the yeast Pumilio homologue Mpt5 recruits Dhh1, a homologue of the Xp54 RNA helicase, indicating that Pumilio-mediated translational repression may involve a similar complex [113]. The mechanism of this highly conserved type of translational repression is not entirely clear, but it is likely to involve assembly of large RNP particles that exclude translation initiation factors and/or ribosomes. In addition, eIF4E1b was shown to be defective in eIF4G binding and to bind 4E-T independently of the canonical eIF4E binding site. This implies that the mRNAs in this complex are translationally repressed through sequestration of the cap by eIF4E1b, which would exclude eIF4E1a/eIF4G from the mRNA. Indeed, tethering of 4E-T causes translational repression and injection of an eIF41b antibody enhances oocyte maturation [143]. It is as yet unclear which proteins in the early complex bind directly to CPEB and therefore are likely to confer specific repression of CPE containing mRNAs. As eIF4E1b is also the predominant CPEB associated eIF4E in fully grown oocytes, this protein could in principle convey CPE-mediated translational repression throughout oogenesis [67,143].
At present it is difficult to choose amongst the multitude of models for translational repression by CPEs. Some of the complexes detected by pulldown and immunoprecipitation may not contain the majority of the repressed mRNA, even though presence of some mRNA was demonstrated by RT-PCR. Alternatively, it may well be that every one of these models is correct at a particular stage of oogenesis or embryogenesis or that the repression is different for specific mRNAs, depending on binding sites for other proteins such as Pumilio and EDEN-BP. An attractive option is that sequestration to P-body like large complexes is the result of translational repression and acts as an enhancer of translational repression, while smaller, more mRNA specific translational repression complexes are formed during the movement of mRNAs in and out of P-bodies. To distinguish between these models, it will be important to characterise the cap binding proteins and P-body components present on specific mRNAs at different developmental stages.
7
Cytoplasmic polyadenylation and translational activation
The simplest explanation for why cytoplasmic polyadenylation leads to translational activation is that the poly(A) tail recruits translation initiation factors through its association with PABP and that these mediate release of the mRNA from the repression complexes, either by exchanging the cap binding complex for eIF4E1a/eIF4G or by somehow extracting the mRNA from a P-body-like RNP complex, a function which has been ascribed to PABP in yeast [150]. In support of this hypothesis, the translational repression of the histone B4 and cyclin B1 3′ UTRs on mRNAs injected into Xenopus oocytes can be abolished by the addition of a poly(A) tail, at least until that tail is deadenylated [29,74]. However other mRNAs (G10, Cl2) appear to be still repressed even if a long poly(A) tail is added and only become translated if the mRNA is actively polyadenylated in the maturing oocyte [6,13]. In a seminal experiment, Barkoff et al. cleaved the endogenous c-Mos mRNA with an oligonucleotide, removing the polyadenylation signal and CPE. As expected, this abrogated c-Mos mRNA polyadenylation and translation in oocytes exposed to progesterone. A ‘prosthetic’ poly(A) tail was tethered to the remaining 3′ UTR using basepairing. This did not lead to c-Mos synthesis in unstimulated oocytes, but restored the synthesis of c-Mos in response to progesterone [76]. This suggests that, at least for the translation of c-Mos mRNA, both the presence of a poly(A) tail and an additional, polyadenylation independent, modification of the mRNP are required for translational activation.
8
Discussion
Since the discovery of CPEB ten years ago, our knowledge of the mechanisms of CPE-mediated cytoplasmic polyadenylation has made great strides, especially in Xenopus oocytes. However, there are still quite a few questions that need to be resolved, for instance:1.A more systematic investigation of the consensus CPE sequence and its maximum distance to the poly(A) signal would enable a more reliable bioinformatic prediction of the targets of CPE-mediated cytoplasmic polyadenylation. Are all mRNAs containing these sequences polyadenylated during oocyte maturation?2.The relative roles of Gld-2 and PAP in cytoplasmic polyadenylation need to be further evaluated. Can they substitute for each other? What are their contact points in the cytoplasmic polyadenylation complex? A cytoplasmic polyadenylation system reconstituted from pure components would be ideal to resolve these questions.3.The kinase(s) responsible for the early activating phosphorylation on Ser174 of CPEB should be identified unequivocally. If Aurora A is responsible, why do inhibitors of its activity not block oocyte maturation and can no early activation of this kinase be detected? Do inhibitors of CamKII affect early polyadenylation? What is the signal transduction cascade leading to induction of the activating kinase?4.What are the relative roles of Musashi and CPEB in the polyadenylation of c-Mos mRNA and other substrates? How do dominant negative mutants of CPEB and Musashi achieve their repressive functions? May there be off target effects through titration of common polyadenylation factors or signal transduction machinery?5.What are the direct interaction between proteins and RNA elements in the cytoplasmic polyadenylation complex? Much of the work discussed here has been performed either in oocyte or reticulocyte lysates, where other interacting proteins may be present. It appears necessary that these interactions are mapped in detail in vitro or in a 2 hybrid system, as this could lead to predictions for the functions of dominant negative mutants and potentially clarify how different complexes are assembled.6.Are the complexes on cytoplasmic polyadenylation sequences other than the CPE similar to the CPEB associated complexes? For instance, do Gld2 or PAP associate with the RNA binding proteins that recognise them?7.Which models for translational repression and deadenylation of CPE containing mRNAs are correct in what stage of oogenesis? Again, a more intimate knowledge of the direct interactions involved in assembling the repression complexes is likely to yield important new investigative tools for resolving this question. In addition, the study of mRNP complexes assembled in vivo could yield some more conclusive answers.8.Are the polyadenylation and translation repression complexes identical for each CPE containing mRNA or are there mRNA specific differences in the complexes? Immunoprecipitation followed by RT-PCR and RNA affinity chromatography can partially answer this question, but affinity purification methods that target specific mRNPs are likely to be crucial to obtain a full answer.
Some of the controversies discussed here are a result of the experimental restrictions of the Xenopus oocyte and egg systems. These systems are very good for biochemical assays both in injected oocytes and egg extracts and they provide ample material for purification and identification. However, the high levels of stable maternal proteins make clean knock down or knock out experiments impossible in most cases, which has led to a heavy reliance on dominant negative approaches. To resolve some of the outstanding issues we are therefore likely to have to look to vertebrate genetic systems such as mouse and/or to develop tissue culture based cytoplasmic polyadenylation systems that are amenable to siRNA knock down, for instance using neuronal cells or cells synchronised in the cell cycle.
The advent of high throughput screens will strongly impact the field of cytoplasmic polyadenylation in the near future as the results of RNP immunoprecipitation microarray experiments and poly(A) tail profiling studies will increase our knowledge of the range of mRNA substrates [9,151–153]. Such screens may also identify new cytoplasmic polyadenylation elements as well as novel cell types and biological processes in which this form of regulation is involved. Only one cytoplasmic polyadenylation element and its binding protein have so far been studied in detail, predominantly in oocyte maturation. As outlined above, a large number of important questions are still unresolved, even in this one system. With at least three other cytoplasmic polyadenylation sequences in existence in oocytes alone and the evidence that cytoplasmic polyadenylation also is involved in neuronal events and the mitotic cell cycle [9], there appears ample scope for expansion of the field in the near future.

translational control
cytoplasmic polyadenylation
oocyte
deadenylation
meiotic maturation


Hippocampal sclerosis dementia differs from hippocampal sclerosis in frontal lobe degeneration

Hippocampal sclerosis (HS) is characterized by selective neuronal loss and gliosis in CA1 and the subiculum and has been associated with several disorders, including Alzheimer’s disease, frontotemporal lobar degeneration with ubiquitin immunoreactive inclusions (FTLD-U), vascular dementia and some tauopathies. In some cases, HS is not associated with other degenerative pathologies. Such cases are sometimes referred to as HS dementia (HSD). Differences between HSD and HS in the setting of FTLD-U have not been systematically investigated. To this end, eight cases of HSD and ten cases of HS associated with FTLD-U were studied with Nissl and periodic acid-Schiff stains to assess neuronal loss and corpora amylacea, respectively. Sections were immunostained with antibodies to glial fibrillary acidic protein, HLA-DR and synaptophysin and immunoreactivity was measured with image analysis in CA1 and the subiculum of each case. Additionally, sections were immunostained with antibodies to 4-R tau to determine the presence of argyrophilic grains. HSD was different from HS associated with FTLD-U. Specifically, it was more common in the elderly, and it was associated with more marked neuronal and synaptic loss and with greater reactive gliosis. Corpora amylacea tended to be more frequent in HSD than in FTLD-U, but there was no difference in frequency of argyrophilic grains.
Introduction
Hippocampal sclerosis (HS) is defined as selective neuronal loss and gliosis of CA1 and the subiculum of the hippocampus. Its prevalence in demented patients ranges from 2.8 to 13% [4–6, 15, 25, 26]. It is associated with several other disorders, including Alzheimer’s disease (AD), frontotemporal lobar degeneration with ubiquitin immunoreactive inclusions (FTLD-U), vascular dementia, dementia with Lewy bodies and some tauopathies. Occasionally, HS has been reported as an independent pathologic explanation for dementia, the so-called HS dementia (HSD). The reported prevalence of HSD ranges from 0.4 to 2% [2, 4, 15, 27].
The etiology of HS is uncertain in most cases, but it is hypothesized to be due to either hypoxic-ischemic injury or to neurodegeneration of selectively vulnerable neuronal populations in CA1 and the subiculum [4, 36]. An hypoxic-ischemic origin is supported by the known deficient hippocampal vascular supply [8] and the fact that CA1 hippocampal neurons have a higher susceptibility to hypoxia due to their concentration of glutamate receptors, which may be involved in excitotoxic cell death [30]. Further arguments in favor of this theory are the association of hypoxic-ischemic episodes in patients with HS [12, 34, 37] and the increased prevalence of cardiac [3, 10, 29] and cerebrovascular disease [11, 12, 26, 35] in cases of HS of presumed hypoxic-ischemic origin.
Since many cases of HS do not have histories of hypoxia or ischemia [2, 10, 12, 15, 27], cardiac disease [2, 11, 12, 27] or cerebrovascular accidents [10, 27], a degenerative cause is also proposed. This hypothesis is supported by the frequent association of HS with degenerative dementias like AD [3, 4, 12, 15, 16, 27, 29] and FTLD-U [3, 4, 13, 15, 19, 22, 24, 29] and the less frequently with tauopathies, mainly argyrophilic grain disease (AGD) and FTDP-17 [5, 12, 17].
The aim of this study was to compare the pathological characteristics of HSD versus HS in the setting of FTD-U.
Materials and methods
The Mayo Clinic Jacksonville brain bank database between 1998 and 2005 of individuals with dementia or degenerative neuropathology (N = 1,487) was screened for specimens with HS that had complete neuropathologic evaluations. This evaluation included description of gross and microscopic findings, as well as quantitative information about Alzheimer type pathology. Specifically, the reports include counts of senile plaques and neurofibrillary tangles in six cortical sections, four sectors of the hippocampus, two regions of the amygdala, as well as subcortical regions with thioflavin-S fluorescent microscopy. It also involves immunohistochemistry for tau, α-synuclein and ubiquitin, as appropriate, as previously described [4]. A Braak neurofibrillary tangle stage was assigned to all cases based upon the distribution of neurofibrillary tangles with thioflavin-S fluorescent microscopy, as previously described [4, 14, 21, 31, 33].
HS was detected in 103 cases; in 95 cases (92%) it was associated with a primary degenerative disease process, mostly AD (36; 18 men and 18 women; 85 ± 5 years of age) and FTLD-U (44; 23 men and 21 women; 75 ± 12 years of age; P < 0.01 compared to AD). HS was detected in about 5% of AD cases (36 of 637) and 75% of FTLD-U cases (44 of 59). The remainder of cases included HS in the setting of a wide range of pathologies, including Lewy body disease, progressive supranuclear palsy, corticobasal degeneration and multiple system atrophy. In eight cases, HS was not associated with degenerative pathology.
For this study, 18 cases were selected for further study and placed in one of two groups. The first group of eight cases was operationally termed HSD; most HSD cases (75%) were associated with evidence of cerebrovascular disease. When compared to a control group of 30 cases from the same brain bank matched for age, sex and Braak stage, cerebrovascular disease was detected in 80% of the controls, a frequency similar to that in HSD. Therefore, we could not necessarily assume that the HS was due to hypoxic-ischemic causes. In all HSD cases, FTLD-U was ruled out with ubiquitin immunohistochemistry. The second group (“FTLD-U HS”) included 10 cases of HS associated to FTLD-U. None of the FTLD-U HS cases had pathologic evidence of cerebrovascular disease or other pathologic processes, and HS was assumed to be related to FTLD.
In all 18 cases other pathologic processes, such as progressive supranuclear palsy, corticobasal degeneration or Lewy body disease were absent. Alzheimer type pathology was minimal. None of the cases in either the HSD or FTLD-U HS group had a Braak neurofibrillary tangle stage greater than IV. In none of the cases could neuronal loss in the hippocampus be attributed to neurofibrillary tangles, since none had many extracellular (“ghost”) neurofibrillary tangles. Medial temporal tauopathy due to AGD was not a basis for exclusion, and all cases were screened for this age-related pathology with immunohistochemistry using isotype-specific tau antibodies, as previously reported [17].
Coronal sections of the posterior hippocampus at the level of the lateral geniculate nucleus were cut at a thickness of 5 μm. Sections were stained with Nissl and periodic acid-Schiff (PAS) stains. Sections were immunostained on a DAKO Autostainer with antibodies to HLA-DR, glial fibrillary acidic protein (GFAP), 4R tau and synaptophysin using procedures similar to those reported previously [23]. The following primary antibodies were used: anti-GFAP (GA-5, monoclonal, 1:1,000, Biogenex, San Ramon, CA), anti-HLA-DR (LN3, monoclonal, 1:100, eBioscience, San Diego, CA), anti-4R tau (ET3, monoclonal, 1:25, Dr. Peter Davies, Albert Einstein College of Medicine, Bronx, NY) and anti-synaptophysin (EP10, monoclonal, 1: 10, Dr. Peter Davies, Albert Einstein College of Medicine, Bronx, NY). For antigen retrieval, slides were steamed in distilled water for 30 min. For ET3 immunostaining, which has been shown to be a sensitive and specific method for detecting AGD [17, 32], slides were also pretreated in 99% formic acid for 30 min.
The degree of neuronal loss in the subiculum, CA1, CA2/3, CA4 (end plate) and dentate gyrus was evaluated on Nissl stained sections using a three-point rating scale: 0, absent; 1, mild-to-moderate; and 2, marked. PAS staining was used to assess the density of corpora amylacea. Corpora amylacea were defined as round, PAS-positive structures in the neuropil, perivascular, subpial and subependymal areas [7]. Presence of corpora amylacea throughout the hippocampus was graded on a four-point scale: 0, absent; 1, sparse; 2, moderate; and 3, many.
To assess astrocytic, microglial and synaptic immunoreactivity, sections immunostained for GFAP, HLA-DR and synaptophysin were used. Standardized, non-overlapping, digital images of the pyramidal layer of CA1 and subiculum were taken under a 40× objective on a light microscope. Immunoreactivity was quantified as a percentage area using MetaMorph software, version 6.3r0 (Molecular Devices, Sunnyvale, CA).
Four-R (4R) tau immunostained slides were used to determine the presence of AGD. Cases were considered to have AGD if they had small dot- or comma-like argyrophilic lesions in neuronal processes of the pyramidal layer and the entorhinal cortex, as well as coiled bodies in temporal white matter, which are the same criteria used in other studies [31].
Clinical and neuropathologic records were reviewed to collect data on age, sex, Braak neurofibrillary tangle stage, brain weight and macroscopic and microscopic neuropathology findings at autopsy. The recorded gross findings at autopsy were lobar, hippocampal and mammillary body atrophy. The remarkable microscopic findings were vascular pathology, ischemic brain injury and white matter disease.
Differences between the two groups in age, Braak stage, brain weight, corpora amylacea, neuronal loss and glial and synaptic immunoreactivity pathology were analyzed with unpaired t test and Mann–Whitney U tests depending upon the variable. Fisher’s exact test was used to compare sex, presence of AGD and remarkable pathologic findings. The statistical analyses were performed using Sigma Stat for Windows, version 3.11 (Systat Software, Richmond, CA) and the significance levels were set at P < 0.05.
Results
A summary of the demographic and neuropathologic findings of the 18 cases included in this study is presented in Table 1. A comparison of the two groups is presented in Table 2. The cases with HSD had an older age of death than those with FTLD-U HS (84 vs. 65, P = 0.001). In the HSD group, six of eight cases were men, while four of six cases in the FTLD-U HS group were men, but this difference was not significant.
Table 1Demographic and neuropathologic features of HS casesAgeSexNFT stageNeuronal lossVascular pathologyCerebrovascular pathologyWMSubCA1CA2CA4DGAtherosclerosisArteriosclerosisAmyloid angiopathyLacunar infarctsMicroscopic infarctsIschemic gliosisP187 MI2200050–75% (PCA)PV WM, BG, Th––F, M, WSAThPVP284 FII22000MinimalBG–BG, ThO–PV, DCP386 M02200050–75% (ICA, MCA)WM, BGF, P, OBG, pons–WM, BG, ThPV, DCP476 MIV02202Minimal–F, O––––P579 MIII1100050–75% (MCA, PCA, ACA)BG, CBO–BGMPVP679 FIII02111––––Cortical WSACortical WSA, ThPVP787 MII22002Minimal–O–––PVP890 MIII21000<50%WM, BG–PV WM, BG, amygdala–ThPV, DCF183 FI22NA00––––––F, T subcorticalF256 M022000––––––F, P subcorticalF356 F021000––––––F, T, P subcorticalF441 FI22002––––––F, T subcorticalF570 MI22000Minimal–––––F, subcorticalF670 M022000––––––F, T subcorticalF762 F010000Minimal–––––F, T subcortical and PVF872 FIII10002––––––F, T subcortical and PVF966 F022002Minimal–––––F, P subcorticalF1073 M022002NA–––––F, T, P subcorticalSub subiculum, CA cornu ammonis, EP endplate, DG dentate gyrus, F frontal, T temporal, P parietal, O occipital, M motor, ICA internal carotid artery, ACA anterior cerebral artery, MCA middle cerebral artery, PCA posterior cerebral artery, VA vertebral artery, BA basilar artery, PV periventricular, DC deep cerebral, WM white matter, BG basal ganglia, Th thalamus, CB cerebellum, WSA watershed area, NA not available, P #  represent HSD cases; F# represent FTLD-U HS casesTable 2Comparison of HSD and FTLD-U HSHSD (n = 8)FTLD-U HS (n = 10)Age at death (years), mean ± Std. Dev.84 ± 565 ± 12‡Sex (% of cases)Male7540Female2560Braak NFT stage, median (25%-tile, 75%-tile)2.25 (1.5, 3)0 (0, 1)†Brain weight (g), mean ± Std. Dev.1130 ± 100868 ± 130‡Gross atrophy (% of cases)Frontal lobe25100†Medial temporal lobe1378†Lateral temporal lobe3367Parietal lobe1767Occipital lobe1311Mammillary bodies100100Hippocampal formation8990Neuronal loss (score), median (25%-tile, 75%-tile)Subiculum1 (0.25, 1)1 (1, 1)CA11 (0.75, 1)1 (0.5, 1)CA2/30 (0, 0.25)0 (0, 0)EP0 (0, 0)0 (0, 0)DG0 (0, 1)0 (0, 1)Synaptophysin (% area), mean ± SEMSubiculum 23.4 ± 12.336.4 ± 21.3CA126.5 ± 24.451.7 ± 20.8†GFAP (% area), mean ± SEMSubiculum 9.5 ± 7.56.2 ± 2.9 CA114.9 ± 12.59.2 ± 7.8HLA-DR (%area), mean ± SEMSubiculum0.80 ± 0.770.56 ± 0.50CA10.59 ± 0.750.19 ± 0.18Corpora amylacea (score), median (25%-tile, 75%-tile)1 (0.5, 1.75)0.5 (0, 2)Argyrophilic grain disease (% of cases)1320Fisher exact test was used for categorical variables, t test for continuous variables and Mann–Whitney Rank Sum test for discontinuous variables†P < 0.05‡P < 0.001
Neuropathologic findings
The Braak neurofibrillary tangle stage was significantly greater in HSD than in FTLD-U HS. The brain weight was also greater in HSD than in FTLD-U HS. Cortical atrophy was more frequent in FTLD-U HS than HSD in frontal (100% vs. 25%), medial temporal (78% vs. 13%), lateral temporal (67% vs. 33%) and parietal lobes (67% vs. 17%). The difference reached statistical significance in frontal and medial temporal regions. Both groups showed marked gross atrophy of the hippocampal formation and mammillary body.
In this comparative study, cerebrovascular pathology was virtually limited to the HSD group. Atherosclerosis of large vessels at the base of the brain was present in seven of eight HSD cases. Three had minimal, 1 had mild (occluding less than 50% of the lumen) and 3 had moderate atherosclerosis (occluding between 50% and 75% of the lumen). Arteriosclerosis defined by presence of hyalinosis of the media and adventitial fibrosis of small (<100 μm diameter) vessels was present in 5 (63%) cases and cerebral amyloid angiopathy (CAA) was present in 4 (50%) of 8 HSD. Three (30%) cases of FTLD-U HS had minimal atherosclerosis. None of the cases of this group had significant arteriolosclerosis or evidence of CAA.
Ischemic brain injury was present in six of eight HSD cases, but in none of the FTLD-U HS cases. The eight HSD cases included 3 (38%) with lacunar infarcts, 4 (50%) with microscopic infarcts and 5 (63%) with microscopic foci of ischemic gliosis.
Most cases, independent of their group, showed white matter pathology defined by rarefaction and attenuation of white matter, associated with dilated perivascular spaces, loss of myelinated fibers and gliosis. These findings were present in the periventricular white matter in seven of eight HSD cases. In three of these cases more diffuse white matter pathology was also present, involving the centrum semiovale. All of the cases with FTLD-U HS showed white matter pathology that followed the distribution of the cortical atrophy.
Two of the HSD cases (P4 and P7) had only minimal atherosclerosis and CAA, which was probably insufficient to cause hippocampal hypoperfusion, but both cases had a history of coronary artery disease and congestive heart failure, which might account for hypoxic-ischemic injury to the hippocampus, although this remains purely speculative, since these risk factors are common in individuals of this age without HS [12].
Another case of HSD (P6) with minimal atherosclerosis, arteriosclerosis and CAA had a history of epilepsy, as well as laminar ischemic gliosis in the watershed cortices and cortical cystic infarcts and thalamic ischemic gliosis. Epilepsy in this patient was probably secondary to a porencephalic cyst that was present in the temporal pole. Given the absence of degenerative pathology, this case was included in the HSD group, but presence of extensive cerebrovascular disease raises the possibility that HS in this case was related to hypoxic-ischemic injury.
Quantitative and semi-quantitative analyses
Both HSD and FTLD-U HS had severe neuronal loss in the pyramidal layer of CA1 and the subiculum. No significant differences were found in the severity of neuronal loss between the two regions; nevertheless, differences in the distribution of neuronal loss were noted. Most cases had neuronal loss in both CA1 and the subiculum, except for two cases of HSD that had isolated CA1 involvement and two cases of FTLD-U HS that had isolated subicular involvement.
The CA2/3 region and the endplate were not affected in most cases. Only cases of HSD occasionally had neuronal loss in these regions, with involvement of CA2/3 in P4 and involvement of CA2/3 and endplate in P6. The dentate was involved in half of the cases of both groups.
Synaptic loss assessed by image analysis of density of synaptophysin immunoreactivity was significantly less in CA1 region in HSD compared to FTLD-U HS (Fig. 1). A similar trend was noted in the subiculum. Gliosis as assessed by image analysis with GFAP and HLA-DR immunostains tended to be greater in HSD compared with FTLD-U HS (Fig. 1), although it did not reach statistical significance probably because of small sample size. Both GFAP and HLA-DR immunoreactivity were greater in HSD than in FTLD-U HS in both CA1 and the subiculum.
Fig. 1Comparison of CA1 region of hippocampus in HS associated with FTLD-U (a, c, e) and HSD (b, d, f) immunostained for synaptophysin (a, b), GFAP (c, d) and HLA-DR (e, f). Note better preservation of neuropil and less gliosis in FTLD-U HS compared to HSD (All figures are ×200)
There tended to be more corpora amylacea in HSD than in FTLD-U HS, but this did not reach statistical significance. A concomitant diagnosis of AGD was made in two cases of FTLD HS and in one case of pure HS (P6).
Discussion
The present study confirmed the characteristic findings of neuronal loss and gliosis in CA1 and the subiculum in HS regardless of etiology; however, there were some significant differences between HSD and HS that occurs in FTLD-U. In addition, there were subjective differences that did not meet statistical significance probably related to the small sample size in this study. The results support the hypothesis that there are differences in underlying disease mechanisms in HSD compared to HS in FTLD-U.
From a demographic perspective, demented patients with HS have been shown to be older than demented patients without HS [11, 12, 27, 29], although there are exceptions [2, 10]. Data concerning the age distribution among patients with HS has to our knowledge not been reported. In the present study, we show HSD to mainly affect the elderly (>80 years of age) as opposed to HS in FTLD-U. The younger age of the FTLD-U may account for the lower frequency of concurrent cerebrovascular disease, since an age-, sex- and Braak neurofibrillary tangle stage-matched cohort had comparable frequency of cerebrovascular disease.
Microscopically, there were three important differences in the pathology in HSD compared with HS in FTLD-U. First, HSD had lower synaptophysin immunoreactivity, suggesting a more destructive process resulting in more severe synaptic loss, while in FTLD-U there is selective neuronal loss, but relative preservation of the neuropil, presumably from preserved afferent and efferent projections to sectors that show neuronal loss. Second, the difference in the distribution of neuronal loss suggested subicular involvement to be characteristic of HS in FTLD-U. Even though the differences in distribution of neuronal loss did not reach statistical significance, other observations support this idea [20]. Third, HSD was associated with greater astrocytic and microglial reaction compared to HS in FTLD-U. Glial activation has been proposed to be caused by neuronal damage, which is present in both, ischemic and degenerative pathology, but may be exacerbated in HSD due to more extensive tissue damage affecting not only neurons, but also the neuropil.
Macroscopically, FTLD-U HS had more severe cortical atrophy, a not surprising finding, since it correlates with FTLD [25]. HSD on the other hand showed a pattern of atrophy often localized to the hippocampal formation, arguing against this pathology being associated with more widespread cortical degeneration. This fits with results from previous studies of HSD that showed that loss of synaptophysin immunoreactivity in the hippocampus was not accompanied with similar loss in the cortex or basal ganglia [12].
Corpora amylacea have been proposed as a possible marker for hypoxic-ischemic HS, since they may be numerous in brains of patients exposed to repetitive hypoxic episodes [1, 6]. They have also been proposed as a useful marker for evaluating HS in surgical specimens removed for treatment of temporal lobe epilepsy [9]. Although there was a trend for more corpora amylacea in HSD than in FTLD-U HS, it did not reach statistical significance, which may be further evidence against the argument that HSD is due to hypoxic-ischemic injury.
Even though we did not find a difference in the frequency of AGD in HSD and HS associated with FTLD-U, the presence of AGD in 3 of the 18 cases, may suggest an association of HS with AGD as reported in previous studies [5, 17].
This study included two cases of HSD (P4 and P7) in which there was not only no evidence of degenerative pathology, but also no evidence of cerebrovascular pathology. Cases in which HS is the only pathologic explanation for dementia are rare. Hattanpa found some cases of HS to be false negative cases for FTLD-U after further studying them with ubiquitin immunohistochemistry [13]. Since we analyzed our cases with ubiquitin, false negatives were excluded, giving a probable explanation for the lower prevalence compared to the 0.48, 0.53 and 2% previously reported by others [2, 15, 27].
The two cases of HSD with neither degenerative nor cerebrovascular pathology had cardiovascular risk factors that could link HS to an ischemic condition of systemic origin. They are similar to the ones that have been reported by others [34, 37]. On the other hand, in a systematic survey of cardiovascular risk factors, including electrocardiograph abnormalities, congestive heart failure and cardiomegaly by imaging and autopsy studies, there was no increased frequency of these findings in HSD compared to age-matched controls without HS [12].
The present study confirms the finding that HS in the setting of dementia in most cases should be regarded as a feature linked to a concurrent degenerative disease process, particularly FTLD-U [13, 18, 19, 28]. In other cases, HS may be linked to a systemic condition (e.g., cardiorespiratory failure) or cerebrovascular disease [8, 36]. In rare cases, the etiology is unknown. The present study suggests that HSD is a disorder of the elderly that is associated with more marked neuronal and synaptic loss and greater reactive gliosis than that seen in HS associated with FTLD-U. This argues for a different pathogenesis

hippocampal sclerosis dementia
image analysis
immunohistochemistry
frontotemporal degeneration


Retracting and seeking movements during laparoscopic goal-oriented movements. Is the shortest path length optimal?

Aims Minimally invasive surgery (MIS) requires a high degree of eye–hand coordination from the surgeon. To facilitate the learning process, objective assessment systems based on analysis of the instruments’ motion are being developed. To investigate the influence of performance on motion characteristics, we examined goal-oriented movements in a box trainer. In general, goal-oriented movements consist of a retracting and a seeking phase, and are, however, not performed via the shortest path length. Therefore, we hypothesized that the shortest path is not an optimal concept in MIS.
Currently, there are various virtual-reality (VR) trainers available to train MIS skills [1, 2]. These trainers have the advantage over box trainers of providing objective feedback about the performance of the user (performance parameters), which motivates residents to learn [3–5]. The most common parameter used to measure basic MIS skills objectively is task completion time. However, this parameter alone does not adequately evaluate the task performance [6]. Therefore, the time for completion is often used in conjunction with other parameters [1, 7–12]. One of the task-independent parameters that is used in each VR trainer is path length – the length of the curve described by the tip of the instrument over time [13]. Intuitively, a longer path length indicates a less-efficient movement and is often interpreted as lack of experience.
In MIS, goal-oriented movements (point-to-point movements) are very common (e.g., during grasping, placing a clip on the vessel, or while using diathermy). In general, a simple strategy to perform such a movement is to move along a straight line between an initial position and a specified target position [14, 15]. However, it is hardly possible to make such a movement during MIS. Movements of MIS instruments are done through the incision point, which acts as a pivot point. Therefore, point-to-point aiming movements in MIS may simply result in a curved workspace path (Fig. 1). Both these ways of performing point-to-point movements are very efficient. In general, however, goal-oriented movements are not performed via the shortest path; a pull-back movement is performed before the specified target position is approached [16, 17]. Therefore, we hypothesized that the shortest path, as presently used to indicate more-efficient performance in MIS, is not a suitable reference.
Fig. 1Three strategies of performing a goal-oriented movement in MIS. (a) a movement along the straight line between the initial and the end position; (b) a movement along a curved path between the initial and the end position, which is the result of the movement of the MIS instrument through the incision point; and (c) a realistic movement performed by the surgeon during the goal-oriented task. A – the initial position; B – the end position; P – incision (pivoting) point
The objective of this study was to examine goal-oriented movements during training of the basic MIS skills. The study consisted of two parts: first, we investigate whether goal-oriented movements can be split into two phases: a retracting phase and a seeking phase; and, subsequently, we investigate whether movements of the MIS instrument in these two phases depend on the surgeon’s experience. The performance of the participants was analyzed and compared using normalized path lengths.
Methods
Participants
Experienced gynaecologists (with the experience of more than 100 MIS procedures), their residents (experience of 10–100 MIS procedures), and novices (medical students with no previous experience in MIS) were invited to participate in this study. Each participant, voluntarily enrolled in this study, was asked to complete a short questionnaire detailing demographic information and prior experience in laparoscopy.
Task
The hypothesis was tested using a simple one-hand positioning task, which requires touching the top of eight cylinders with the tip of the MIS instrument (Fig. 2). All cylinders were situated in a box trainer in various three-dimensional (3D) positions. Every correctly touched cylinder resulted in the lighting of the lamp corresponding to this cylinder. The start/end point and the order of touching cylinders (indicated by the numbers located next to the cylinders) were the same for each participant.
Fig. 2The positioning task. The task requires touching the top of the eight cylinders (of varying 3D position) with the tip of the MIS instrument. A correctly touched cylinder results in lighting up a lamp (above on the picture) corresponding to this cylinder. The start/end point and the order of touching the cylinders are indicated by the numbers located next to the cylinders
To mimic the in vivo gynaecological MIS situation, all participants performed the task with their left hand, while the camera was held in the right hand. To provide the same conditions for each participant, the position of the task and the incision points for the camera and the MIS instrument were standardized in the box trainer. The image of a 0° laparoscope was presented on a monitor.
Before the test, all participants were instructed how to perform the positioning task. Furthermore, they were allowed to make one trial before testing.
Data analysis
Movements of the MIS instrument were recorded with our newly developed TrEndo tracking system with a sample frequency of 100 Hz [18]. The data of the eight simple goal-oriented movements performed by each participant during the task were analyzed. The first goal-oriented movement was defined by the movement between the start point and first cylinder. Each successive goal-oriented movement was defined by the movement between two consecutive cylinders (Fig. 2).
The retracting phase describes the pull-back movement of the MIS instrument in the direction of the pivoting point. Therefore, we analyzed the projected movements of the instrument in the ABP plane (Fig. 3), which passes through the begin point of the movement (point A), the end point of the movement (point B), and the pivoting point (point P). To examine the deviation of the movement from the plane ABP, we also analyzed the movements projected in the plane AB, which is perpendicular to the plane ABP, and which passes through the points A and B (Fig. 3). Point M, defined as the point of the movement most distant from the line AB, was used to make a distinction between the retracting phase AM (from point A to point M) and the seeking phase MB (from point M to point B). The analysis of each of the two phases was done using a normalized path length, which was derived as follows:  where PLreal is the real path length (between A and M for retracting, and M and B for seeking phase), and PL0 is the distance (length of the straight line) between A and M and M and B (for retracting, and seeking phases, respectively). The normalized path lengths of all eight successive goal-oriented movements were averaged per participant and analyzed.
Fig. 3The ABP (described by the points A, B, and P) and AB (that passes through points A and B, and is perpendicular to the plane ABP) planes presented in an orthographic projection: (a) the ABP and AB planes, and the goal-oriented movement presented in the three-dimensional space; (b) front view: projection of the goal-oriented movement in the ABP plane; (c) top view: projection of the goal-oriented movement in the AB plane; and (d) side view: the ABP and AB planes, and the goal-oriented movement presented in a side view. A – the initial position; B – the end position; M – a point, which is used to make a distinction between the retracting and the seeking phases; P – incision (pivoting) point
Statistics
Data were analyzed using the Statistics Toolbox of MATLAB 7. Statistical analysis was performed using one-way analysis of variance (ANOVA), and Wilcoxon tests. A probability p < 0.05 was considered to be statistically significant.
Results
Participants
Thirty-eight participants completed the task and the questionnaire. The group of experts consisted of 9 experts (age 38–59 years). The group of residents consisted of 17 gynaecological residents (age 29–41 years). The group of novices consisted of 12 medical students (age 23–28 years). All participants were right-handed.
Retracting and seeking phases
Typical trajectories for an expert and a novice performing the goal-oriented movement are presented in Fig. 4. The figure shows that the retracting and seeking phases can easily be distinguished from each other. No significant difference between the experts, residents, and novices was observed in the distance between point M and the line AB.
Fig. 4The typical trajectories for an expert (left) and a novice (right) performing a goal-oriented movement. Top: projection of the goal-oriented movement in the plane ABP. Bottom: projection of the goal-oriented movement in the plane AB. A – the initial position; B – the end position; M – a point, which is used to make a distinction between the retracting and the seeking phases
Normalized path lengths of the two phases are presented in Fig. 5. In 3D space, the experts’ normalized path length was shorter during the retracting phase [median (range) %: 152 (129–178)] than during the seeking phase [180 (172–247)]. Similarly, the residents’ and novices’ path lengths were shorter during the retracting phase [164 (126–250), and 168 (136–268)] than during the seeking phase [201 (163–287), and 290 (244–469), respectively].
Fig. 5Normalized path lengths of the retracting and the seeking phases for experts, residents, and novices. Upper: normalized path length in 3D space. Middle: normalized path length in the ABP plane. Lower: normalized path length in the AB plane. The results are presented as notched box-and-whisker plots, where every box has a line at every quartile, median, and upper quartile values. The whiskers are presented as lines that extend from each end of the box in order to show the extent of the rest of the data. The notches represent the 95% confidence interval for the median. Boxes whose notches do not overlap are significantly different (p < 0.05). **p < 0.01; ***p < 0.001; E – experts; R – residents; N – novices; 1 – retracting phase; 2 – seeking phase
In the ABP plane, the experts’ normalized path length in the retracting phase [132 (120–194)] was significantly shorter than in the seeking phase [199 (154–234)]. The normalized path lengths of residents and novices were also significantly shorter in the retracting phase [133 (108–492), and 180 (118–287)] than in the seeking phase [208 (162–286), and 310 (252–469)].
In the AB plane, the experts’ normalized path length was significantly shorter in the retracting phase [122 (100–200)] than in the seeking phase [183 (141–209)]. Normalized path lengths of residents and novices were also significantly shorter during the retracting phase [147 (107–264), and 207 (128–271)] than during the seeking phase [199 (162–285), and 310 (252–469)].
Influence of the experience
The experts’, residents’, and novices’ normalized path lengths during the retracting and the seeking phases are presented in Fig. 6. The data in Fig. 6 are the same as the data in Fig. 5, but presented per phase for all groups. In 3D space, there was no significant difference between experts, residents, and novices for the retracting phase. The normalized path lengths of experts and residents were significantly shorter than the path length of the novices during the seeking phase. No significant difference between experts and residents was observed in that phase.
Fig. 6The influence of the experience on the performance of the retracting and the seeking phases. The data presented in this figure are the same as the data in Fig. 7, but presented per phase. *p < 0.05; ***p < 0.001; E – experts; R – residents; N – novices; 1 – retracting phase; 2 – seeking phase
In the ABP plane, we found no significant difference between experts, residents, and novices for the retracting phase. In the seeking phase, the normalized path lengths of experts and residents were significantly shorter than the path length of novices. No significant difference between experts and residents was observed in that phase.
In the AB plane, a significant difference between experts, residents, and novices was found for both phases. In both the retracting and the seeking phases, the normalized path lengths of the experts and residents were significantly shorter than the path length of the novices. No significant difference between experts and residents was observed for both phases.
Discussion
Our findings indicate that goal-oriented movements are not performed via the shortest path. The movements clearly distinguish a retracting and a seeking phase. The results show that the normalized path length during the retracting phase is significantly shorter than during the seeking phase. Furthermore, experience in MIS does only influence the seeking phase. Experts’ and residents’ normalized path lengths were significantly shorter than the path lengths of the novices. The shorter path length in the seeking phase implies better performance; therefore, the seeking phase is characteristic of the differences in performance. This finding confirms that the path length is an important measure in the assessment of basic MIS skills, in which the seeking phase is the only component that can distinguish between novice and expert.
The retracting phase is essential in MIS, because it improves safety of the patient by avoiding unpredicted contact with the tissue. It seems that the best strategy to perform this phase safely is to pull back the instrument along its axis (in the direction of the pivoting point) and avoid any movements in the AB plane, which can cause unpredicted contacts with the tissue. The results of this study confirm this assumption; experts’ and residents’ normalized path lengths are smaller in the AB plane than the path length of the novices. Movements in the AB plane can result in unintended tissue contact and be dangerous for the patient; therefore, it would be beneficial to let novices learn how to perform a more-precise retracting movement.
The experience in MIS does not influence the retracting phase in the ABP plane. Moreover, a longer retraction of the MIS instrument in the direction of the pivoting point does not denote worse or less-efficient performance. In contrast, it may be a sign of the safer (for the patient) use of the MIS instruments. This finding is important, because it shows that the shorter path length, as presently used to indicate more-efficient performance in MIS, is not a correct concept for analyzing optimal movements in laparoscopy.
To investigate whether the retracting phase really represents safety, it is necessary to design a different study. For example, one possibility would be to include obstacles which, upon touching represent an error. With such a study design, it would be feasible to determine how much retraction is necessary and whether the experts have learnt the optimal retraction amount. The present study cannot be used to decide on these aspects.
The results of this study indicate that the comparison of the novices’ path length to the experts’ path length is an important and valid component of the overall criterion-based assessment of basic MIS skills. It is necessary to realize that this study was not designed to determine whether expert performance actually results in the optimal path length and, therefore, the results of this study cannot be used to indicate that experts’ movements are optimal.
In the literature, there is a tendency to assess MIS performance using very basic terms (e.g., time, path length, penalty points), which do not show “the size and the nature of the gap between expert and novice performance” [13]. The results of this study show that the analysis of the separate phases of the movement in various planes can give such details. For example, movements of the MIS instrument along its axis (in and out the trocar) during the seeking phase characterize the surgeon’s level of depth perception. Movements of the instrument in the AB plane may indicate eye–hand coordination problems (e.g., orientation). Since every phase of the movement analyzed in different planes provides details about different limitations, it is crucial to analyze the movements in MIS separately for each phase and in both planes. Only then will the feedback about the performance and the nature of the limitations lead to goal-oriented training curricula.
This study shows for the first time the importance of proper analysis of the instruments’ movements during training of MIS skills. The clinical impact of such extended analysis is that only in this way is it possible to implement a correct objective score that will measure and certify the competence of surgeons’ basic psychomotor MIS skills in addition to the existing criteria for the assessment of MIS performance. Moreover, this extended motion analysis can result in improvement of the training of basic MIS skills, since it will identify the differences between the experts’ and novices’ performance, and which areas require more training.
Conclusions
Movements during laparoscopic tasks are not performed via the shortest path. Therefore, the shortest path length, as presently used for the assessment of basic MIS skills, may be not a proper concept for analyzing optimal movements and therefore needs to be revised. Goal-oriented movements should be split into two phases: a retracting and a seeking phase. Novices are less efficient in the seeking phase. This finding confirms that path length is an important measure in the assessment of basic MIS skills, but that the seeking phase is the only component that distinguishes between novice and expert. Furthermore, the retracting phase is very important in MIS, since it improves safety by avoiding intermediate tissue contact. Analyzing motions in the separate phases provides greater insight into the nature of the gap between expert and novice performance. Motion analysis in MIS should be seen as an addition to the existing criteria in assessing performance.

goal-oriented movement
path length
minimally invasive surgery
objective assessment
training
motion analysis