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Currently, domestic production of vegetable soybean (aka “edamame”) lags well behind consumer demand, with approximately 70% of U.S.-consumed edamame imported each year. A major barrier for growth of the U.S. edamame industry is an overall lack of varieties with adequate consumer acceptability and adaption to the U.S. climate and environment. In this study, we evaluated eleven vegetable soybean genotypes (including one commercial check) for differences in yield, pod size, and resistance to local insect, bacterial, and fungal pressures in order to identify genotypes with the greatest potential for use in commercial edamame production. Although there were variations in average pod length (42.1 - 53.6 mm), width (10.9 - 12.7 mm), and thickness (6.29 - 7.34 mm) among the genotypes, only pod length showed statistical significance. In addition, genotype significantly affected fresh pod yield. The prevalence of specific insect pests varied by location and year and included soybean aphid, potato leafhopper, Mexican bean beetle, as well as a complex of stink bugs and lepidopteran larvae. For each of these insect pests, significant differences were observed. Some plant diseases observed on the edamame genotypes included: downy mildew, bacterial pustule, Fusarium pod rot, Cercospora blight and purple seed stain, and damping off. In 2018, in Whitethorne, VA, soybean downy mildew was quite prevalent and disease symptoms varied considerably. Overall, genotypes V16-0524 and R15-10280 showed particularly favorable yield, and resilience to native pests compared to the commercial check, UA-Kirksey. The genotypes V16-0524 and R15-10280 showed strong potential to increase the availability of varieties that can be used for commercial edamame production in the Mid-Atlantic region.

Intraflagellar transport (IFT) carries proteins into flagella but how IFT trains interact with the large number of diverse proteins required to assemble flagella remains largely unknown. Here, we show that IFT of radial spokes in Chlamydomonas requires ARMC2/PF27, a conserved armadillo repeat protein associated with male infertility and reduced lung function. Chlamydomonas ARMC2 was highly enriched in growing flagella and tagged ARMC2 and the spoke protein RSP3 comigrated on anterograde trains. In contrast, a cargo and an adapter of inner and outer dynein arms moved independently of ARMC2, indicating that unrelated cargoes distribute stochastically onto the IFT trains. After concomitant unloading at the flagellar tip, RSP3 attached to the axoneme whereas ARMC2 diffused back to the cell body. In armc2/pf27 mutants, IFT of radial spokes was abolished and the presence of radial spokes was limited to the proximal region of flagella. We conclude that ARMC2 is a cargo adapter required for IFT of radial spokes to ensure their assembly along flagella. ARMC2 belongs to a growing class of cargo-specific adapters that enable flagellar transport of preassembled axonemal substructures by IFT.

Brown marmorated stink bug, Halyomorpha halys (Stål), is an invasive species in the United States that attacks a wide variety of agricultural commodities including fruits, vegetables, agronomic crops, and ornamental plants. Populations of H. halys adults were collected from four and six states in 2017 and 2018, respectively, and tested using topical applications to establish baseline levels of susceptibility to two commonly used insecticides, bifenthrin and thiamethoxam. A Probit-estimated (95% fiducial limits) LD50 and LD99 of 2.64 g AI/L (1.2–3.84 g AI/L) and 84.96 g AI/L (35.76–716.16 g AI/L) for bifenthrin, and a LD50 and LD99 of 0.05 g AI/liter (1.14E-5–0.27 g AI/L) and 150.11 g AI/L (27.35–761,867 g AI/L) for thiamethoxam, respectively. These baseline levels can be used for future insecticide resistance monitoring in H. halys.

We determined how the ciliary motor I1 dynein is transported. A specialized adapter, IDA3, facilitates I1 dynein attachment to the ciliary transporter called intraflagellar transport (IFT). Loading of IDA3 and I1 dynein on IFT is regulated by ciliary length., Axonemal dyneins, including inner dynein arm I1, assemble in the cytoplasm prior to transport into cilia by intraflagellar transport (IFT). How I1 dynein interacts with IFT is not understood. We take advantage of the Chlamydomonas reinhardtii ida3 mutant, which assembles the inner arm I1 dynein complex in the cytoplasm but fails to transport I1 into the cilium, resulting in I1 dynein-deficient axonemes with abnormal motility. The IDA3 gene encodes an ∼115-kDa coiled-coil protein that primarily enters the cilium during ciliary growth but is not an axonemal protein. During growth, IDA3, along with I1 dynein, is transported by anterograde IFT to the tip of the cilium. At the tip, IDA3 uncouples from IFT and diffuses within the cilium. IFT transport of IDA3 decreases as cilia lengthen and subsides once full length is achieved. IDA3 is the first example of an essential and selective IFT adapter that is regulated by ciliary length.

Corn rootworm remains the key pest of maize in the United States. It is managed largely by Bt corn hybrids, along with soil insecticides and neonicotinoid seed treatments (NSTs), the latter of which are applied to virtually all conventionally (non-Bt) produced maize. Frequently, more than one of these pest-management approaches is employed at the same time. To determine the utility and relative contributions of these various approaches, a meta-analysis was conducted on plant health and pest damage metrics from 15 yr of insecticide efficacy trials conducted on Indiana maize to compare the pest-protection potential of NSTs to that of other insecticides and Bt hybrids. The probability of recovering the insecticide cost associated with each treatment was also calculated when possible. With the exception of early-season plant health (stand counts), in which the NSTs performed better than all other insecticides, the vast majority of insecticides performed similarly in all plant health metrics, including yield. Furthermore, all tested insecticides (including NSTs) reported a high probability (>80%) of recovering treatment costs. Given the similarity in performance and probability of recovering treatment costs, we suggest NSTs be optional for producers, so that they can be incorporated into an insecticide rotation when managing for corn rootworm, the primary Indiana corn pest. This approach could simultaneously reduce costs to growers, lower the likelihood of nontarget effects, and reduce the risk of pests evolving resistance to the neonicotinoid insecticides.

A lack of target specificity has greatly hindered the success of inhibitor development against matrix metalloproteinases (MMPs) for the treatment of various cancers. The MMP catalytic domains are highly conserved, whereas the hemopexin-like domains of MMPs are unique to each family member. The hemopexin-like domain of MMP-9 enhances cancer cell migration through self-interaction and heterointeractions with cell surface proteins including CD44 and α4β1 integrin. These interactions activate EGFR-MAP kinase dependent signaling that leads to cell migration. In this work, we generated a library of compounds, based on hit molecule N-[4-(difluoromethoxy)phenyl]-2-[(4-oxo-6-propyl-1H-pyrimidin-2-yl)sulfanyl]-acetamide, that target the hemopexin-like domain of MMP-9. We identify N-(4-fluorophenyl)-4-(4-oxo-3,4,5,6,7,8-hexahydroquinazolin-2-ylthio)butanamide, 3c, as a potent lead (Kd = 320 nM) that is specific for binding to the proMMP-9 hemopexin-like domain. We demonstrate that 3c disruption of MMP-9 homodimerization prevents association of proMMP-9 with both α4β1 integrin and CD44 and results in the dissociation of EGFR. This disruption results in decreased phosphorylation of Src and its downstream target proteins focal adhesion kinase (FAK) and paxillin (PAX), which are implicated in promoting tumor cell growth, migration, and invasion. Using a chicken chorioallantoic membrane in vivo assay, we demonstrate that 500 nM 3c blocks cancer cell invasion of the basement membrane and reduces angiogenesis. In conclusion, we present a mechanism of action for 3c whereby targeting the hemopexin domain results in decreased cancer cell migration through simultaneous disruption of α4β1 integrin and EGFR signaling pathways, thereby preventing signaling bypass. Targeting through the hemopexin-like domain is a powerful approach to antimetastatic drug development.

Flea beetles, are common pests of cabbage Brassica oleracea L. (Brassicales: Brassicaceae) and eggplant Solanum melongena L. (Solanales: Solanaceae), but little is known about the flea beetle populations in Virginia, their impact on yield, or the most effective control methods. This research investigates flea beetle populations and the impact of their feeding injury on cabbage and eggplant in Southwest Virginia and determines the most efficacious control methods. In Whitethorne, VA, cabbage and eggplant crops were vacuum sampled weekly throughout two summers (2015, 2016). Crucifer flea beetle, Phyllotreta cruciferae (Goeze) (Coleoptera: Chrysomelidae), and striped flea beetle, Phyllotreta striolata Fabr. (Coleoptera: Chrysomelidae) were found on cabbage; whereas, eggplant flea beetle, Epitrix fucula (Crotch) (Coleoptera: Chrysomelidae), and the tobacco flea beetle, Epitrix hirtipennis (Melsheimer) (Coleoptera: Chrysomelidae) were found on eggplant. To evaluate the impact of flea beetle feeding on these plants flea beetle densities and defoliation were assessed weekly and individual plant, as well as whole plot yields, assessed at harvest. For cabbage, significant yield reductions were observed between 1 and 20% and >60% defoliation. Similarly, significant yield reductions were observed between 41 and 60% and >60% defoliation for eggplant. The efficacy of various insecticides was also evaluated. Soil application of the systemic neonicotinoid dinotefuran, imidacloprid, and the foliar-applied bifenthrin resulted in the fewest beetles, the least amount of leaf defoliation, and the highest yield in cabbage and eggplant. This research helps vegetable growers to better understand the severity of these pests and how to effectively combat them.

Spotted knapweed, Centaurea stoebe L. (Asteraceae), is an invasive perennial forb that has become economically and ecologically damaging in North America. The weevil Larinus minutus Gyllenhal (Coleoptera: Curculionidae), a biological control agent of invasive knapweeds, was introduced to 37 sites in northwest Arkansas since 2008 as part of a biological control program for spotted knapweed. In 2011 and 2012, 25 of these release sites were surveyed to monitor how L. minutus infestation rates changed in relation to distance and time from release. The initial L. minutus introductions at these sites occurred from 2008 to 2011. Transects were used from the point of initial weevil introduction to establish sampling quadrats in which capitula were collected to document weevil presence and infestation rates. The mean maximum distance of weevil colonization and mean local abundance (within the first 50 m from the release point) were calculated in relation to time (yr) since release. Five sites had > 10 quadrats in both sampling years and were analyzed with an exponential decay function to model localized population growth and spread. Annual increases in mean local abundance and maximum distance of colonization were observed. These results were substantiated by localized growth (at 3 sites) and spread (at 2 sites) that occurred at the 5 sites analyzed with the exponential decay function. These findings suggest that in years following L. minutus introduction, assuming similar release strategies and environmental conditions, consistent increases in weevil infestation rates and spread from the release site may be expected in about half the sites at which populations establish. USDA-APHIS-PPQUnited States Department of Agriculture (USDA) We thank Edward Gbur Jr. and Fred Stephen for their input on experimental design, and Elizabeth Long for her feedback on the manuscript. This work was supported in part through a Cooperative Agreement with USDA-APHIS-PPQ.

We developed quantitative assays to test the hypothesis that the N-DRC is required for integrity of the ciliary axoneme. We examined reactivated motility of demembranated drc cells, commonly termed "reactivated cell models." ATP-induced reactivation of wild-type cells resulted in the forward swimming of ∼90% of cell models. ATP-induced reactivation failed in a subset of drc cell models, despite forward motility in live drc cells. Dark-field light microscopic observations of drc cell models revealed various degrees of axonemal splaying. In contrast, >98% of axonemes from wild-type reactivated cell models remained intact. The sup-pf4 and drc3 mutants, unlike other drc mutants, retain most of the N-DRC linker that interconnects outer doublet microtubules. Reactivated sup-pf4 and drc3 cell models displayed nearly wild-type levels of forward motility. Thus, the N-DRC linker is required for axonemal integrity. We also examined reactivated motility and axoneme integrity in mutants defective in tubulin polyglutamylation. ATP-induced reactivation resulted in forward swimming of >75% of tpg cell models. Analysis of double mutants defective in tubulin polyglutamylation and different regions of the N-DRC indicate B-tubule polyglutamylation and the distal lobe of the linker region are both important for axonemal integrity and normal N-DRC function. © 2016 Wiley Periodicals, Inc.

Radial spokes are conserved macromolecular complexes that are essential for ciliary motility. Little is known about the assembly and functions of the three individual radial spokes, RS1, RS2, and RS3. In Tetrahymena, a conserved ciliary protein, FAP206, docks RS2 and dynein c to the doublet microtubule., Radial spokes are conserved macromolecular complexes that are essential for ciliary motility. A triplet of three radial spokes, RS1, RS2, and RS3, repeats every 96 nm along the doublet microtubules. Each spoke has a distinct base that docks to the doublet and is linked to different inner dynein arms. Little is known about the assembly and functions of individual radial spokes. A knockout of the conserved ciliary protein FAP206 in the ciliate Tetrahymena resulted in slow cell motility. Cryo–electron tomography showed that in the absence of FAP206, the 96-nm repeats lacked RS2 and dynein c. Occasionally, RS2 assembled but lacked both the front prong of its microtubule base and dynein c, whose tail is attached to the front prong. Overexpressed GFP-FAP206 decorated nonciliary microtubules in vivo. Thus FAP206 is likely part of the front prong and docks RS2 and dynein c to the microtubule.

Cytoplasmic assembly of ciliary dyneins, a process known as preassembly, requires numerous non-dynein proteins, but the identities and functions of these proteins are not fully elucidated. Here, we show that the classical Chlamydomonas motility mutant pf23 is defective in the Chlamydomonas homolog of DYX1C1. The pf23 mutant has a 494 bp deletion in the DYX1C1 gene and expresses a shorter DYX1C1 protein in the cytoplasm. Structural analyses, using cryo-ET, reveal that pf23 axonemes lack most of the inner dynein arms. Spectral counting confirms that DYX1C1 is essential for the assembly of the majority of ciliary inner dynein arms (IDA) as well as a fraction of the outer dynein arms (ODA). A C-terminal truncation of DYX1C1 shows a reduction in a subset of these ciliary IDAs. Sucrose gradients of cytoplasmic extracts show that preassembled ciliary dyneins are reduced compared to wild-type, which suggests an important role in dynein complex stability. The role of PF23/DYX1C1 remains unknown, but we suggest that DYX1C1 could provide a scaffold for macromolecular assembly., Author summary Most animal cells have antenna-like organelles called “cilia”. These organelles have various important functions both in motility and sensing the environment. Motile cilia are essential for moving cells as well as moving fluids across a surface. The waveform of motile cilia requires large macromolecular motors; these are the ciliary dyneins. These dynein complexes are assembled in the cytoplasm in a pathway called preassembly, and then transported into cilia. Defects in this process cause a heterogeneous human disease called primary ciliary dyskinesia that results, for example, in the disruption of the motility of respiratory tract cilia, sperm and nodal cilia during development. The mechanisms of the preassembly pathway are not fully understood. In this study, we use a mutation in the well-conserved DYX1C1/PF23 gene of the green alga, Chlamydomonas reinhardtii. Loss of a conserved domain (DYX) reveals a failure to assemble most ciliary dyneins. Preassembly of inner arm dyneins is particularly affected. We find that if dynein arms are not assembled, dynein subunits in the cytoplasm are unstable. We suggest that DYX1C1 may play a role as a scaffold for other preassembly factors and the dynein subunits.

Neonicotinoid seed treatments, typically clothianidin or thiamethoxam, are routinely applied to >80% of maize (corn) seed grown in North America where they are marketed as a targeted pesticide delivery system. Despite this widespread use, the amount of compound translocated into plant tissue from the initial seed treatment to provide protection has not been reported. Our two year field study compared concentrations of clothianidin seed treatments in maize to that of maize without neonicotinoid seed treatments and found neonicotinoids present in root tissues up to 34 days post planting. Plant-bound clothianidin concentrations followed an exponential decay pattern with initially high values followed by a rapid decrease within the first ~20 days post planting. A maximum of 1.34% of the initial seed treatment was successfully recovered from plant tissues in both study years and a maximum of 0.26% was recovered from root tissue. Our findings show neonicotinoid seed treatments may provide protection from some early season secondary maize pests. However, the proportion of the neonicotinoid seed treatment clothianidin translocated into plant tissues throughout the growing season is low overall and this observation may provide a mechanism to explain reports of inconsistent efficacy of this pest management approach and increasing detections of environmental neonicotinoids.

To determine mechanisms of assembly of ciliary dyneins, we focused on the Chlamydomonas inner dynein arm, I1 dynein, also known as dynein f. I1 dynein assembles in the cytoplasm as a 20S complex similar to the 20S I1 dynein complex isolated from the axoneme. The intermediate chain subunit, IC140 (IDA7), and heavy chains (IDA1, IDA2) are required for 20S I1 dynein preassembly in the cytoplasm. Unlike I1 dynein derived from the axoneme, the cytoplasmic 20S I1 complex will not rebind I1-deficient axonemes in vitro. To test the hypothesis that I1 dynein is transported to the distal tip of the cilia for assembly in the axoneme, we performed cytoplasmic complementation in dikaryons formed between wild-type and I1 dynein mutant cells. Rescue of I1 dynein assembly in mutant cilia occurred first at the distal tip and then proceeded toward the proximal axoneme. Notably, in contrast to other combinations, I1 dynein assembly was significantly delayed in dikaryons formed between ida7 and ida3. Furthermore, rescue of I1 dynein assembly required new protein synthesis in the ida7 × ida3 dikaryons. On the basis of the additional observations, we postulate that IDA3 is required for 20S I1 dynein transport. Cytoplasmic complementation in dikaryons using the conditional kinesin-2 mutant, fla10-1 revealed that transport of I1 dynein is dependent on kinesin-2 activity. Thus, I1 dynein complex assembly depends upon IFT for transport to the ciliary distal tip prior to docking in the axoneme.

To address the mechanisms of ciliary radial spoke assembly, we took advantage of the Chlamydomonas pf27 mutant. The radial spokes that assemble in pf27 are localized to the proximal quarter of the axoneme, but otherwise are fully assembled into 20S radial spoke complexes competent to bind spokeless axonemes in vitro. Thus, pf27 is not defective in radial spoke assembly or docking to the axoneme. Rather, our results suggest that pf27 is defective in the transport of spoke complexes. During ciliary regeneration in pf27, radial spoke assembly occurs asynchronously from other axonemal components. In contrast, during ciliary regeneration in wild-type Chlamydomonas, radial spokes and other axonemal components assemble concurrently as the axoneme grows. Complementation in temporary dikaryons between wild-type and pf27 reveals rescue of radial spoke assembly that begins at the distal tip, allowing further assembly to proceed from tip to base of the axoneme. Notably, rescued assembly of radial spokes occurred independently of the established proximal radial spokes in pf27 axonemes in dikaryons. These results reveal that 20S radial spokes can assemble proximally in the pf27 cilium but as the cilium lengthens, spoke assembly requires transport. We postulate that PF27 encodes an adaptor or modifier protein required for radial spoke – IFT interaction.

In protostomes, cell polarity is present after fertilization whereas most deuterostome embryos show minimal polarity during the early cleavages. We now show establishment of cell polarity as early as the first cleavage division in sea urchin embryos. We find, using the apical markers G M1 , integrins, and the aPKC–PAR6 complex, that cells are polarized upon insertion of distinct basolateral membrane at the first division. This early apical–basolateral polarity, similar to that found in much larger cleaving amphibian zygotes, reflects precocious functional epithelial cell polarity. Isolated cleavage blastomeres exhibit polarized actin-dependent fluid phase endocytosis only on the G M1 , integrin, microvillus-containing apical surface. A role for a functional PAR complex in cleavage plane determination was shown with experiments interfering with aPKC activity, which results in several spindle defects and compromised blastula development. These studies suggest that cell and embryonic polarity is established at the first cleavage, mediated in part by the Par complex of proteins, and is achieved by directed insertion of basolateral membrane in the cleavage furrow.

Chlamydomonas reinhardtii is an outstanding model genetic organism for study of assembly of cilia. Here, methods are described for synchronization of ciliary regeneration in Chlamydomonas to analyze the sequence in which ciliary proteins assemble. In addition, the methods described allow analysis of the mechanisms involved in regulation of ciliary length, the proteins required for ciliary assembly, and the temporal expression of genes encoding ciliary proteins. Ultimately, these methods can contribute to discovery of conserved genes that when defective lead to abnormal ciliary assembly and human disease.

Cell migration is a critical determinant of cancer invasion and metastasis. Drugs targeting cancer cell migration have been hindered due to the lack of effective assays for monitoring cancer cell migration. Here we describe a novel method to microscopically monitor cell migration in a quantitative fashion. This assay can be used to study genes involved in cancer cell migration, as well as screening anticancer drugs that target this cellular process.

Amyloid-beta peptides (Abeta) are widely presumed to play a causal role in Alzheimer disease. Release of Abeta from the amyloid precursor protein (APP) requires proteolysis by the beta-site APP-cleaving enzyme (BACE1). Although increased BACE1 activity in Alzheimer disease brains and human (h) BACE1 transgenic (tg) mice results in altered APP cleavage, the contribution of these molecular alterations to neurodegeneration is unclear. We therefore used the murine Thy1 promoter to express high levels of hBACE1, with or without hAPP, in neurons of tg mice. Compared with hAPP mice, hBACE1/hAPP doubly tg mice had increased levels of APP C-terminal fragments (C89, C83) and decreased levels of full-length APP and Abeta. In contrast to non-tg controls and hAPP mice, hBACE1 mice and hBACE1/hAPP mice showed degeneration of neurons in the neocortex and hippocampus and degradation of myelin. Neurological deficits were also more severe in hBACE1 and hBACE1/hAPP mice than in hAPP mice. These results demonstrate that high levels of BACE1 activity are sufficient to elicit neurodegeneration and neurological decline in vivo. This pathogenic pathway involves the accumulation of APP C-terminal fragments but does not depend on increased production of human Abeta. Thus, inhibiting BACE1 may block not only Abeta-dependent but also Abeta-independent pathogenic mechanisms.

Irrigated field experiments were conducted near Torrington, WY, during the 2001 to 2002 (year 1) and 2002 to 2003 (year 2) winter wheat growing seasons to evaluate cultivar response to different imazamox rates, adjuvants, and application timings. Five cultivars were treated postemergence in the early fall (EF), late fall (LF), or early spring (ES) with imazamox at 54 or 108 g ai/ha, including either nonionic surfactant (NIS) at 0.25% or methylated seed oil (MSO) at 1% (v/v) as adjuvants. A 28% urea ammonium nitrate solution at 1% (v/v) was included with all treatments. Spring injury was more severe in year 1 than year 2. Severe spring injury on ‘AP502 CL’, ‘Above’, ‘IMI-Fidel’, ‘IMI-Jagger’, and ‘IMI-Madsen’ was linked to fall application of 108 g/ha imazamox with MSO. Imazamox applied at 108 g/ha plus MSO applied in the fall consistently injured all cultivars more than the same rate with NIS and 54 g/ha imazamox regardless of adjuvant and timing, although severity of injury in the experiments differed between EF and LF timings in years 1 and 2, respectively. Correlation analysis supports injury reduced reproductive tillers per meter of row and wheat yields and increased the number of seeds per spike in year 1. The reduction of reproductive tillers per meter of row in year 1 was likely the result of severe injury caused by 108 g/ha imazamox applied in the EF coupled with little snow cover to protect against cold winter temperatures. Wheat yield in year 1 was reduced by 108 g/ha imazamox applied in the early fall; however, imazamox applied at 54 g/ha with either adjuvant in EF, LF, or ES were safe. Yield parameters and wheat yields in year 2 were not affected by imazamox rate, adjuvant, timing, or interactions of these factors.

Field experiments were conducted at five locations in Kansas, Nebraska, and Wyoming to determine the effects of imazamox rate and application timing on winter annual grass control and crop response in imidazolinone-tolerant winter wheat. Imazamox at 35, 44, or 53 g ai/ha applied early-fall postemergence (EFP), late-fall postemergence, early-spring postemergence (ESP), or late-spring postemergence (LSP) controlled jointed goatgrass at least 95% in all experiments. Feral rye control with imazamox was 95 to 99%, regardless of rate or application timing at Hays, KS, in 2001. Feral rye control at Sidney, NE, and Torrington, WY, was highest (78 to 85%) with imazamox at 44 or 53 g/ha. At Sidney and Torrington, feral rye control was greatest when imazamox was applied EFP. Imazamox stunted wheat

Background: The neurodegenerative process in Alzheimer’s disease (AD) and in the Lewy body variant of AD (LBV) patients is characterized by cholinergic dysfunction and deposition of amyloid β-peptide (Aβ) 1–40 and 1–42; however, the differential effects of Aβ species on the cholinergic system are not completely clear. Objective: To better understand the relationship between levels of Aβ1–40 and 1–42 on cholinergic deficits in AD and LBV patients. Methods: Levels of choline acetyltransferase (ChAT) activity and ChAT immunoreactivity in the plaques in the frontal cortex of patients with AD and LBV were correlated with Aβ1–42 and 1–40 levels determined by ELISA and with neuropathologic and neurologic markers. Results: Although the overall levels of ChAT activity were reduced in AD and LBV cases, there was a direct correlation with Aβ1–42 levels. Furthermore, patients with high Aβ1–42 levels had more abundant cholinergic dystrophic neurites in the plaques than cases with lower Aβ1–42. Conclusion: Aβ1–42 may also trigger cholinergic dysfunction by promoting aberrant neuritic sprouting.

Both monoamine oxidase (MAO) A and MAO B in the brain have been implicated in the etiology of Alzheimer's disease. MAO B is elevated in plaque-associated glia in Alzheimer brain. Elevations in MAO A in Alzheimer neurons have been linked to increases in neurotoxic metabolites and neuron loss. We investigated the relationship between cognitive function in Alzheimer patients and post-mortem prefrontal cortex MAO A and B activities. Prefrontal cortex tissue from 92 Alzheimer patients and 74 neurologically normal subjects was obtained at autopsy and analyzed for activities of MAO A and B by radioenzymatic methods. Mini Mental Status Exam was performed on Alzheimer patients within 1 year of death. Alzheimer brains were analyzed for Braak stage, tangles, plaques and choline acetyltransferase activity. Prefrontal cortex MAO B activity was significantly increased by 16% in Alzheimer patients versus normals, whereas MAO A activity was significantly decreased by 17% in these same patients. Neither MAO A nor MAO B activities correlated with cognitive function (MMSE score), choline acetyltransferase activity, plaques, neurofibrillary tangles, Braak stage, or age of disease onset in the Alzheimer patients. With increasing Alzheimer duration or increasing Braak stage, MMSE scores and choline acetyltransferase activity declined, but levels of MAO A and B in prefrontal cortex were unchanged. Patients in the upper quintile for MAO A or B activity did not differ significantly from those in the lowest quintile with respect to MMSE scores or age of Alzheimer disease onset. We conclude that the changes in MAO A and B in the prefrontal cortex occur very early in Alzheimer's disease and remain relatively constant as the disease progresses.

Apolipoprotein E (apoE) is known to bind to at least five receptors, including the low-density lipoprotein (LDL) receptor-related protein (LRP), very low density LDL receptor (VLDL-R), LDL-R, apoE receptor 2 (apoER2), and megalin/gp330. In this context, the main objective of the present study was to better understand the contributions of LRP and LDL-R to the in vivo neurotrophic effects of apoE. For this purpose, apoE-deficient and receptor-associated protein (RAP)-deficient mice were infused with recombinant apoE3, RAP, or saline. Infusion of apoE3 into apoE-deficient mice resulted in amelioration of degenerative alterations of pyramidal neurons, but had no effect on somatostatin-producing interneurons. In contrast, infusion of apoE3 into RAP-deficient mice resulted in amelioration of degenerative alterations of somatostatin-producing interneurons. LRP and LDL-R levels were significantly reduced in RAP-deficient mice, but significantly increased in the apoE-deficient mice. In contrast, levels of apoE were reduced in the RAP-deficient mice compared to wildtype controls, suggesting that neurotrophic effects of apoE3 in the RAP-deficient mice were related to a combined deficit in endogenous apoE and selected apoE receptors. Furthermore, in apoE-deficient mice, infusion of apoE3 had a neurotrophic effect on somatostatin-producing interneurons only when combined with RAP, suggesting that increased expression of apoE receptors in apoE-deficient mice prevented apoE from rescuing somatostatin-producing neurons. This study supports the contention that some of the in vivo neurotrophic effects of apoE are mediated by LRP and LDL-R and that a critical balance between levels of apoE and its receptors is necessary for the differential neurotrophic effects to appear.

Cell shape and membrane remodeling rely on regulated interactions between the lipid bilayer and cytoskeletal arrays at the cell cortex. During cytokinesis, animal cells build an actomyosin ring anchored to the plasma membrane at the equatorial cortex. Ring constriction coupled to plasma membrane ingression separates the two daughter cells [1]. Plasma-membrane lipids influence membrane biophysical properties such as membrane curvature and elasticity and play an active role in cell function [2], and specialized membrane domains are emerging as important factors in regulating assembly and rearrangement of the cytoskeleton [3]. Here we show that mutations in the gene bond [4], which encodes a Drosophila member of the family of Elovl proteins that mediate elongation of very-long-chain fatty acids [5], block or dramatically slow cleavage-furrow ingression during early telophase in dividing spermatocytes. In bond-mutant cells at late stages of division, the contractile ring frequently detaches from the cortex and constricts or collapses to one side of the cell, and the cleavage furrow regresses. Our findings implicate very-long-chain fatty acids or their derivative complex lipids in allowing supple membrane deformation and the stable connection of cortical contractile components to the plasma membrane during cell division.

Recent studies have shown that deficient functioning of glutamate transporters (GTs) in Alzheimer disease (AD) might lead to neurodegeneration via excitotoxicity; however, the characteristics of cell death and pathways involved are not yet clear. The main objective of the present study was to determine if deficient GT functioning in AD could be associated with cell damage and caspase activation. For this purpose, we analyzed the levels of caspase-1 and 3 immunoreactivity in AD and control brains and correlated this data with the numbers of cells displaying DNA fragmentation, GT activity, and amyloid precursor protein (APP) mRNA expression. Compared to controls, AD cases showed extensive positive labeling of neurons and glial cells with an assay for DNA fragmentation suggestive of cell damage, as well as increased neuronal caspase-3 and Bcl-2 immunoreactivity. Linear regression analysis showed a strong negative correlation between GT activity and apoptosis, and between deficient GT functioning and caspase-3 immunoreactivity. Neurons displaying DNA fragmentation presented more intense caspase-3 immunoreactivity than intact neurons. In addition, the altered ratio between the spliced forms of APP correlated with DNA fragmentation and caspase-3 immunolabeling. Taken together, these results support the possibility that excitotoxic injury associated with deficient GT functioning and an imbalance in ratio of spliced APP forms might lead to cell death via caspase-3 activation.

The loss of neocortical synapses that occurs in Alzheimer's disease (AD) has been shown to correlate with cognitive decline. In addition, marked losses in the cholinergic system in AD, specifically choline acetyltransferase (ChAT) activity and high affinity presynaptic neuronal nicotinic cholinergic receptors (nAChRs), have also been described. We hypothesized that in AD, the loss of [3H]-ligand binding to nAChRs, which are largely presynaptic, would correlate with changes in two other presynaptic markers: synaptophysin (Syn), a measure of synaptic density, and ChAT activity. The midfrontal (MF) cortex of 36 autopsy confirmed (NIA and CERAD criteria) AD patients (mean death age +/- SD 80.1 +/- 8.4 years) who met NINDS-ADRDA criteria for a clinical diagnosis of probable or possible AD, and 11 nondemented controls (mean death age +/- SD 77.9 +/- 8.0) were examined. Synapse counts were quantified by a dotimmunobinding assay for Syn. ChAT activity was assessed by standard biochemical assays. Nicotinic cholinergic receptor binding was assayed using the high affinity nicotinic agonist [3H]-(+/-)-epibatidine ([3H]-EPI). The mean +/- SD Syn in AD (83.4 +/- 31.9 arbitrary units (AU)/mg protein) was significantly lower than controls (126.1 +/- 19.9, p = 0.0003; t-test). The mean ChAT activity in AD (139.0 +/- 75.6 nmol ACh/hr/100 mg protein) was significantly lower than controls (219.6 +/- 70.8, p = 0.004). The mean [3H]-EPI total binding in AD (6.2 +/- 2.8 fmol/mg protein) was significantly lower than controls (14.8 +/- 3.2; p < 0.0001). Syn correlated with [3H]-EPI binding in AD (r = 0.48, p = 0.006; Pearson) but ChAT did not (r = -0.20, p = 0.34). We conclude that loss of high affinity nAChR binding correlates with loss of synapses in AD. The lack of correlation between [3H]-EPI binding and ChAT activity suggests that the targeted receptor populations may not be located exclusively on cholinergic neurons.

Excitatory neurotransmitters such as glutamate are required for the normal functioning of the central nervous system but can trigger excitotoxic neuronal injury if allowed to accumulate to abnormally high levels. Their extracellular levels are controlled primarily by transmitter uptake into astrocytes. Here, we demonstrate that the amyloid protein precursor may participate in the regulation of this important process. The amyloid protein precursor has been well conserved through evolution, and a number of studies indicate that it may function as an endogenous excitoprotectant. However, the mechanisms underlying this neuroprotective capacity remain largely unknown. At moderate levels of expression, human amyloid protein precursors increased glutamate/aspartate uptake in brains of transgenic mice, with the 751-amino acid isoform showing greater potency than the 695-amino acid isoform. Cerebral glutamate/aspartate transporter protein levels were higher in transgenic mice than in non-transgenic controls, whereas transporter mRNA levels were unchanged. Amyloid protein precursor-dependent stimulation of aspartate uptake by cultured primary astrocytes was associated with increases in protein kinase A and C activity and could be blocked by inhibitors of these kinases. The stimulation of astroglial excitatory amino acid transport by amyloid protein precursors could protect the brain against excitotoxicity and may play an important role in neurotransmission.

Vesicle trafficking and new membrane addition at the cleavage furrow have been extensively documented. However, less clear is the old idea that expansion at the cell poles occurs during cytokinesis. We find that new membrane is added to the cell poles during anaphase, causing the plasma membrane to expand coincident with the constriction of the contractile ring and may provide a pushing force for membrane ingression at the furrow. This membrane addition occurs earlier during mitosis than membrane addition at the furrow and is dependent on actin and astral microtubules. The membrane that is added at the polar regions is compositionally distinct from the original cell membrane in that it is devoid of GM1, a component of lipid rafts. These findings suggest that the growth of the plasma membrane at the cell poles during cell division is not due to stretching as previously thought, but due to the addition of compositionally unique new membrane. © 2012 Wiley Periodicals, Inc

The amyloid precursor protein (APP) is involved in Alzheimer's disease (AD) because its degradation products accumulate abnormally in AD brains and APP mutations are associated with early onset AD. However, its role in health and disease appears to be complex, with different APP derivatives showing either neurotoxic or neurotrophic effects in vitro. To elucidate the effects APP has on the brain in vivo, cDNAs encoding different forms of human APP (hAPP) were placed downstream of the neuron-specific enolase (NSE) promoter. In multiple lines of NSE-hAPP transgenic mice neuronal overexpression of hAPP was accompanied by an increase in the number of synaptophysin immunoreactive (SYN-IR) presynaptic terminals and in the expression of the growth-associated marker GAP-43. In lines expressing moderate levels of hAPP751 or hAPP695, this effect was more prominent in homozygous than in heterozygous transgenic mice. In contrast, a line with several-fold higher levels of hAPP695 expression showed less increase in SYN-IR presynaptic terminals per amount of hAPP expressed than the lower expressor lines and a decrease in synaptotrophic effects in homozygous compared with heterozygous offspring. Transgenic mice (2–24 months of age) showed no evidence for amyloid deposits or neurodegeneration. These findings suggest that APP may be important for the formation/maintenance of synapses in vivo and that its synaptotrophic effects may be critically dependent on the expression levels of different APP isoforms. Alterations in APP expression, processing or function could contribute to the synaptic pathology seen in AD.

Recent evidence has revealed that the dynein motors and highly conserved signaling proteins are localized within the ciliary 9 + 2 axoneme. One key mechanism for regulation of motility is phosphorylation. Here, we review diverse evidence, from multiple experimental organisms, that ciliary motility is regulated by phosphorylation / dephosphorylation of the dynein arms through kinases and phosphatases that are anchored immediately adjacent to their axonemal substrates.

About one quarter of Alzheimer's disease patients have been found to have concomitant subcortical and neocortical Lewy bodies (LBs). We compared the aberrant neuronal sprouting and the extent of neuritic and synaptic damage in these Lewy body variant of Alzheimer's disease (LBV), with the same pathologic alterations in Alzheimer's disease without LBs (AD). More of the thioflavine-S-positive senile plaques of the LBVs contained growth associated protein 43 (GAP-43), a marker of neuritic growth and sprouting. Compared to AD, the LBVs had 39% more GAP-43-positive plaques in the frontal cortex, and 53% more in the hippocampus. These neuritic alterations were accompanied by an accumulation of amyloid precursor protein and phosphorylate neurofilaments.. Synapse loss was the same in LBV and AD. These results suggest more extensive aberrant neuronal sprouting in LBV than in AD.

The expression levels of three synaptic proteins (synaptophysin, synaptotagmin, and growth-associated protein 43 [GAP43]) in AD cases clinically classified by Clinical Dementia Rating (CDR) score were analyzed. Compared with control subjects (CDR = 0), mild (early) AD (CDR = 0.5 to 1) cases had a 25% loss of synaptophysin immunoreactivity. Levels of synaptotagmin and GAP43 were unchanged in mild AD, but cases with CDR of >1 had a progressive decrement in these synaptic proteins. Thus, synaptic injury in frontal cortex is an early event in AD.

Both protein kinases and phosphoprotein phosphatases are important components of signal transduction systems in cells. Recent studies in Alzheimer's disease (AD) have shown abnormal protein phosphorylation in the cortex suggesting an alteration in these enzymes. In the present study, an antibody against CD45 was used to analyze the status of this protein phosphotyrosine phosphatase in AD. We studied and quantified the immunohistochemical and immunochemical distribution of this integral membrane protein in control and AD brain. We found that anti-CD45 immunostained the great majority of microglia, both resting and activated. These cells were Ricinus communis agglutinin I positive and glial fibrillary acidic protein and neurofilament negative. The AD frontal cortex showed a 35% (P less than 0.01) increase in the number of anti-CD45 immunoreactive microglia as compared with controls. These results were consistent with the immunoblot quantification of CD45 immunoreactivity following native gel electrophoresis. In AD, 30% of the CD45-immunostained microglia were clustered in the neuritic plaques (about six per plaque) while the remaining 70% were scattered in the neuropil. The AD hippocampus showed an increase in CD45-immunoreactive microglia in the molecular layer of the dentate gyrus. At the ultrastructural level, CD45 immunoreactivity was localized exclusively to the plasma membrane of the microglia. The presence of the anti-CD45 immunoreactivity in microglia suggests the possibility that they may require the presence of CD45 as a cell surface receptor which may regulate cell function through modulation of intracellular signaling.

Currently available specific synaptic markers have made it possible to estimate the synaptic density by immunochemical techniques. In the present study we labeled the neocortical presynaptic terminals in histological sections of human autopsy tissue with a monoclonal antibody against synaptophysin. The characteristic granular neuropil reaction was quantified by measuring the average optical density (OD) in the different layers of the parietal cortex with the aid of image analysis equipment. The raw neuropil OD was corrected by subtracting the OD of the white matter in the same section. Our study showed that consistent microdensitometric results can be obtained on 5-microns paraffin sections from specimens with less than 8 hr of post-mortem time before fixation, incubated with 5 micrograms/ml of anti-synaptophysin. The corrected OD measurements were slightly larger in neocortical layers II, III, and V than in layers I, IV, and VI, but the differences were not statistically significant. In area 17, layer IV was denser than the others. We conclude that with certain precautions this method can be used to measure relative amounts of synaptophysin-like immunoreactivity and to infer the density of presynaptic boutons in human situations and in animal models.

Signaling pathways within the mitotic mechanism temporally orchestrate spindle assembly with chromosome capture and alignment, and then coordinate initiation of chromosome segregation with spindle breakdown and cytokinesis for reproductive success. Kinetochore localized Mad2p acts in the spindle assembly checkpoint pathway during prophase and prometaphase to monitor bipolar attachment of chromosomes to spindle microtubules as well as proper tension at kinetochores. Once established, Mad2p is not degraded, but instead transits to spindle poles preceding the metaphase/anaphase transition in human and yeast cells. Whether conserved relocalization of Mad2p to poles is a final step in the spindle assembly checkpoint pathway or whether the post-metaphase transition allows Mad2p to cooperate in anaphase events leading to mitotic exit has been unknown. We examined post-metaphase localization of Mad2p in fission yeast. Our observations indicate an extended signaling pathway for Mad2p that includes kinetochore to bipolar localization at spindle poles, then additional transitions from bipolar to unipolar to equatorial. We determined that Mad2p associates with the microtubule organizing center complex through direct binding to Alp4p and that microtubule motor proteins Kinesin-14 Pkl1 and Dynein contribute to Mad2p anaphase transitions. At anaphase B onset, bipolar to unipolar transitions of both Mad2p and the septation inititiation network (SIN) kinase Cdc7 are observed. We determined that Mad2p and Cdc7p transitions monitor different events in anaphase, but that neither are required for anaphase B initiation. Our findings indicate that altered Mad2p anaphase spindle localizations can reflect changes in spindle function during mitotic exit that could contribute to fidelity in anaphase events.

The low-density lipoprotein receptor-related protein (LRP) is an abundant neuronal cell surface receptor that regulates amyloid beta-protein (Abeta) trafficking into the cell. Specifically, LRP binds secreted Abeta complexes and mediates its degradation. Previously, we have shown in vitro that the uptake of Abeta mediated by LRP is protective and that blocking this receptor significantly enhances neurotoxicity. To further characterize the effects of LRP and other lipoprotein receptors on Abeta deposition, an in vivo model of decreased LRP expression, receptor-associated protein (RAP)-deficient (RAP-/-) mice was crossed with human amyloid protein precursor transgenic (hAPP tg) mice, and plaque formation and neurodegeneration were analyzed. We found that, although the age of onset for plaque formation was the same in hAPP tg and hAPP tg/RAP-/- mice, the amount of amyloid deposited doubled in the hAPP tg/RAP-/- background. Moreover, these mice displayed increased neuronal damage and astrogliosis. Together, these results further support the contention that LRP and other lipoprotein receptors might be neuroprotective against Abeta toxicity and that this receptor might play an integral role in Abeta clearance.

We investigated the potential mechanisms through which Cerebrolysin™, a neuroprotective noothropic agent, might affect Alzheimer’s disease pathology. Transgenic (tg) mice expressing mutant human (h) amyloid precursor protein 751 (APP751) cDNA under the Thy-1 promoter (mThyl-hAPP751) were treated for four weeks with this compound and analyzed by confocal microscopy to asses its effects on amyloid plaque formation and neurodegeneration. In this model, amyloid plaques in the brain are found much earlier (beginning at 3 months) than in other tg models. Quantitative computer-aided analysis with anti-amyloid-β protein (Aβ) antibodies, revealed that Cerebrolysin significantly reduced the amyloid burden in the frontal cortex of 5-month-old mice. Furthermore, Cerebrolysin treatment reduced the levels of Aβ1–42. This was accompanied by amelioration of the synaptic alterations in the frontal cortex of mThyl-hAPP751 tg mice. In conclusion, the present study supports the possibility that Cerebrolysin might have neuroprotective effects by decreasing the production of Af and reducing amyloid deposition.

Objective: To determine the timing of cholinergic loss and reduction of synapses in AD. Background: Decrements in neocortical synapses and cholinergic function occur in AD and correlate with cognitive decline. However, how early in the disease process these changes appear remains unclear. Methods: An autopsy series of 89 demented patients with pathologically confirmed AD (National Institute on Aging and Consortium to Establish a Registry for Alzheimer’s Disease criteria) and 18 normal control subjects (NC). The AD cases were stratified according to their last Mini-Mental State Examination (MMSE) score prior to death as mild (MMSE = 20; n = 14), moderate (MMSE = 10 to 19; n = 20), severe (MMSE = 1 to 9; n = 29), and very severe (MMSE = 0; n = 26). Midfrontal (MF) synapse density was assessed by dot-immunobinding assay for synaptophysin (Syn), and MF choline acetyltransferase (ChAT) activity was determined using standard protocols. Results: Compared with those in NC, neither Syn nor ChAT was appreciably reduced in patients with mild AD at death. Decline of ChAT was significant only in AD patients who died in the late stages of the disease and was maximal in those who had more severely deteriorated. In contrast, decline of Syn was significant and almost maximal in patients in intermediate or moderate stages. Consequently, the last MMSE score prior to death correlated more strongly with ChAT than Syn when the AD cohort included more impaired patients ( r = 0.46 versus 0.40). The reverse occurred when only less impaired patients (MMSE = 10) were included in the analyses ( r = 0.28 versus 0.64). There was only a modest correlation between Syn and ChAT activity. Conclusions: The results imply an asynchronous pattern of decline of synapses and cholinergic activity, with Syn loss preceding ChAT decrements. However, neither MF synapse reduction nor cholinergic dysfunction appears to be an early event in AD.

Recent studies have shown that amyloid precursor protein (APP), which plays a central role in Alzheimer's disease (AD), protects against excitotoxic neuronal injuries by regulating the function of the glial glutamate transporters. The mechanisms underlying these effects and their relationship to the neurodegenerative process in AD are under intense scrutiny. In this context, the main objective of the present study was to determine if overexpression of mutant human APP in transgenic mouse brains results in altered functioning of the excitatory amino acid transporters (EAATs). Transgenic mice expressing the 695 amino acid form of the human APP from the Thy-1 promoter showed a significant decrease in B(max) and K(D) for aspartate uptake when compared to nontransgenic controls. This decrease in glutamate transporter activity was associated with decreased protein expression of glial specific glutamate transporters, EAAT1 and 2, but did not affect mRNA levels. These results suggest that expression of mutant forms of APP disturbs astroglial transport of excitatory amino acids at the posttranscriptional level leading, in turn, to increased susceptibility to glutamate toxicity.

Objective: To investigate the relationship between apolipoprotein E ( APOE ) genotype and both cholinergic dysfunction and synapse loss in AD. Background: A reduction in neocortical synapses and marked losses in the cholinergic system occur in AD. It has been suggested that the number of APOE e4 alleles is inversely related to choline acetyltransferase (ChAT) activity, thereby influencing cholinergic function. Whether APOE genotype may influence neocortical synapse loss remains unclear. Methods: An autopsy series of 182 patients with AD (National Institute on Aging and Consortium to Establish a Registry for Alzheimer’s Disease criteria) and 16 normal controls (NC). APOE genotype was determined in blood samples or in postmortem brain tissue. Midfrontal synapse counts (AU/μg) were quantified by a dot-immunobinding assay for synaptophysin (Syn). Midfrontal ChAT activity (nmol/h/100 mg) was assessed using standard assays. Results: Mean midfrontal ChAT activity and Syn were both significantly reduced in patients with AD compared with NC. The relationship between ChAT activity and number of e4 allele copies in AD was complex, with ChAT activity lower in patients with either two or no e4 alleles compared with those with one e4 allele. There was no relationship between APOE genotype and synapse loss in AD. Syn density was almost identical across the three genotypes. Conclusions: Unlike other studies, we failed to detect a linear relationship between ChAT activity and number of e4 allele copies in the midfrontal cortex of this large sample of patients with AD. Our data also show that the presence of e4 allele does not influence midfrontal synapse loss in AD. This suggests that factors other than APOE genotype may be operative in the decline in midfrontal cholinergic function and synapses seen in AD.

Recent studies have suggested that neuronal populations that contain glutamate receptors are vulnerable to damage mediated by the human immunodeficiency virus 1 (HIV-1). Somatostatin-immunoreactive neurons contain, among other elements, glutamate receptors, and might therefore be susceptible to HIV-mediated damage. In order to test this hypothesis, we compared patterns of somatostatin immunoreactivity in the cortex and subcortex of autopsied AIDS cases with and without HIV encephalitis (HIVE). Somatostatin immunoreactivity in the frontal cortex interneurons, hippocampal pyramidal and nonpyramidal cells, and globus pallidus was significantly reduced in HIVE. Radioimmunoassay demonstrated a comparable decrease in somatostatin levels in the neocortex of HIVE cases. The decrease in somatostatin immunoreactivity in the neocortex was inversely correlated with the severity of HIVE and global cognitive performance, but not with the extent of the astroglial reaction. These findings indicate that somatostatin-immunoreactive neurons in the cortex are susceptible to damage mediated by HIV and that deficient functioning of this neuronal population might contribute to the cognitive dysfunction observed in AIDS patients.

Synaptic loss and neurofibrillary pathology are major contributors to the cognitive deficits in Alzheimer’s disease (AD), indicating an altered connectivity of association neurocircuitries. Synaptic damage occurs early in the development of AD, suggesting that synapse pathology is a primary rather than a secondary event. The mechanisms of synaptic damage and neurodegeneration in AD are not completely understood. Recent studies have suggested that abnormal expression and/or processing of growth-associated proteins in the central nervous system might play a role in the mechanisms leading to synaptic damage and neurodegeneration in AD. Prominent among these proteins are amyloid precursor protein (APP), apolipoprotein E (apoE), and non Aβ amyloid component (NAC) precursor (NACP). All of these molecules have several common features: 1) modulation of synaptic function, 2) involvement in amyloidogenesis, and 3) mutations (APP) and polymorphisms (APOE, NACP) that are associated with a higher risk for AD. Abnormal functioning of synaptic-related proteins with amyloidogenic potential might play a central role in the pathogenesis of AD. In this context, the main objectives of this manuscript are to review the contribution of synaptic alterations to the mechanisms of dementia in AD and to discuss some of the possible mechanisms through which malfunctioning of APP, apoE and NACP might lead to synaptic damage and plaque formation in AD.

The mechanisms of synapse damage in Alzheimer's disease (AD) are not fully understood. Deficient functioning of glutamate transporters might be involved in synaptic pathology and neurodegeneration by failing to clear excess glutamate at the synaptic cleft. In AD, glutamate transporter activity as assessed by D-[3H]aspartate binding is decreased; however, it is not clear to what extent it is associated with the neurodegenerative process and cognitive alterations. For this purpose, levels of D- and L-[3H]aspartate binding in midfrontal cortex were correlated with synaptophysin levels, brain spectrin degradation product levels, and clinical and neuropathological indicators of AD. Compared to control brains, AD brains displayed a 34% decrease in levels of D-[3H]aspartate binding, a 30% decrease in L-[3H]aspartate binding, and a 48% loss of synaptophysin immunoreactivity. Increased levels of brain spectrin degradation products correlated with a decrease in levels of D-[3H] and L-[3H]aspartate binding, and decreased levels of synaptophysin immunoreactivity. Levels of L-[3H]aspartate binding correlated with levels of synaptophysin immunoreactivity. These results suggest that decreased glutamate transporter activity in AD is associated with increased excitotoxicity and neurodegeneration, supporting the possibility that abnormal functioning of this system might be involved in the pathogenesis of synaptic damage in AD.

Apolipoprotein E (apoE) is involved in the development and regeneration of the central nervous system (CNS). Recent studies have shown that the expres sion of allele e4 of APOE is a major risk factor for Alzheimer’s disease (AD), a neurodegenerative disorder associated with synaptic loss, amyloid deposition and disruption of the neuronal cytoskeleton. In order to better understand the mechanisms by which apoE is involved in AD, it is necessary to understand the physiological role of this molecule in the intact CNS. For this purpose, we studied the fate of synaptic and dendritic populations during aging and after injury to the CNS in apoE-deficient (knockout) mice. In apoE deficient homozygous mice, there was an age-dependent 15%–40% loss of synaptophysin-immunoreactive nerve terminals and microtubule-associated protein 2-immunoreactive dendrites in the neocortex and hippocampus, when compared to controls. Dendritic alterations were observed as early as 4 months of age. Ultrastructural analysis revealed extensive dendritic vacuolization and disruption of the endomembrane system and cytoskeleton in apoE-deficient homozygous mice. The dendritic damage was accompanied by a decreased immunoreactivity for cytoskeletal markers α and β tubulin (but not kinesin) in the cell bodies and processes of neurons of apoE-deficient mice. In addition, apoE-deficient homozygous mice showed a delay in the patterns of reinnervation of the outer molecular layer of the hippocampal dentate gyrus after perforant pathway transection, and in the neocortex after intraperitoneal kainic acid injection. These results support the contention that apoE might play an important role in maintaining the stability of the synapto-dendritic apparatus, and that altered or deficient functioning of this molecule could underlie the synaptic and cytoskeletal alterations observed in AD.

Apolipoprotein E (apoE) is involved in the development and regeneration of the central nervous system (CNS). ApoE may also be necessary to maintain the integrity of the synapto-dendritic complexity. We analyzed the synaptic alterations in the CNS of apoE-deficient (knockout) mice during the aging process. In apoE-deficient homozygous mice, there was an age-dependent 15 to 40% loss of synaptophysin-immunoreactive nerve terminals and microtubule-associated protein 2-immunoreactive dendrites in the neocortex and hippocampus, when compared to controls. Dendritic alterations were observed as early as 4 months of age. Ultrastructural analysis revealed extensive dendritic vacuolization and disruption of the endomembrane system and cytoskeleton in apoE-deficient homozygous mice. Further immunocytochemical studies of the neuronal cytoskeleton showed that in apoE-deficient mice there was a decrease in the immunoreactivity of alpha and beta tubulin (but not kinesin) in the cell bodies and processes. These results support the contention that apoE might play an important role in maintaining the stability of the synapto-dendritic apparatus and that altered or deficient functioning of this molecule could underlie the synaptic and cytoskeletal alterations in Alzheimer's disease.

Both genetic and environmental factors affect the progression of Alzheimer's disease (AD). The presence of cortical Lewy bodies in AD patients is associated with an altered presentation of AD pathology suggestive of an interaction between the pathogenesis of Lewy bodies and AD lesions. Since the CYP2D6B mutant allele is often present in patients with Lewy body diseases (Parkinson's disease and Lewy body variant of AD), we extended these prior observations by studying the neuropathology associated with the presence of the CYP2D6B mutant allele in a pure AD population without Lewy bodies. AD patients who possessed the CYP2D6B mutant allele, in comparison with those without the CYP2D6B allele, were found to have a smaller decline in two synaptic markers, choline acetyltransferase and synaptophysin, in the frontal cortex relative to normal control values. On the other hand, senile plaques and neurofibrillary tangles were not significantly affected by the presence of the CYP2D6B mutant allele in the frontal cortex of AD patients. Association of the CYP2D6B mutant allele with Lewy body formation in both Parkinson's disease and the Lewy body variant of AD and with the milder synaptic pathology in pure AD without Lewy bodies suggest that depending on the contribution of other genetic and environmental factors, this mutant allele may be involved with different aspects of neurodegeneration.

In the present study we observed that while platelet-derived growth factor (PDGF)-BB is exclusively expressed by neurons in the human brain, PDGF-AA is expressed in neurons and blood vessels. In Alzheimer's disease (AD), antibodies against PDGF-BB (but not PDGF-AA) recognized the neurofibrillary alterations of this disease. The levels of PDGF-BB correlated with the patterns of synaptic loss and sprouting while PDGF-AA immunostaining of the vessels was correlated with glial proliferation. Immunostaining was completely abolished when the antibodies were preincubated with their respective purified recombinant PDGF. Western blot analysis showed that antibodies against PDGF recognized a 31 kDa protein that was mildly increased in AD. These data suggest that PDGF, as well as other neurotrophic factors, play an important role in the mechanisms of neurofibrillary pathology in AD.

Expression of the HIV-1 envelope protein gp120 in brains of transgenic (tg) mice induces extensive neurodegeneration (Toggas, S. M., E. Masliah, E. M. Rockenstein, G. F. Rall, C. R. Abraham, and L. Mucke. 1994. Nature [Lond.]. 367:188-193.). To further analyze the pathogenesis of gp120-induced neurotoxicity and to assess the neuroprotective potential of human amyloid precursor proteins (hAPPs) in vivo, different hAPP isoforms were expressed in neurons of gp120/hAPP-bigenic mice: hAPP751, which contains a Kunitz-type protease inhibitor domain, or hAPP695, which lacks this domain. Bigenic mice overexpressing hAPP751 at moderate levels showed significantly less neuronal loss, synapto-dendritic degeneration, and gliosis than singly tg mice expressing gp120 alone. In contrast, higher levels of hAPP695 expression in bigenic mice failed to prevent gp120-induced brain damage. These data indicate that hAPP can exert important neuroprotective functions in vivo and that the efficiency of this protection may depend on the hAPP isoform expressed and/or on the level of neuronal hAPP expression. Hence, molecules that mimic beneficial APP activities may be useful in the prevention/treatment of HIV-1-associated nervous system damage and, perhaps, also of other types of neural injury.

Double stranded (ds) RNA activated protein kinase PKR was discovered over 30 years ago as a downstream mediator of the interferon signal pathway. Initial studies focused on PKR's anti-virus and pro-inflammatory effects. More recently, PKR has been found to be involved in signal transduction pathways critical for the negative regulation of cell growth and initiation of apoptosis. Significantly, loss of PKR expression/activity has been associated with increased growth of human breast carcinoma, B-cell CLL, and T-cell ALL using patient samples. Furthermore, PKR activity is increased in bone marrow progenitor cells isolated from patients with myelodysplastic syndrome (MDS) which undergo significant apoptosis compared to normal marrow progenitors not observed when evolved to AML. Thus, in the process of MDS evolution to leukemia, PKR expression/activity may be downregulated by a process that is currently unclear. To explore the role of PKR in MDS/AML we have constructed a transgenic mouse model expressing human PKR (TgPKR) under control of vav regulatory elements to drive expression specifically in hematopoietic-tissue. We hypothesized transgenic expression of human PKR would exert an inhibitory effect on mouse hematopoiesis, cause ineffective blood cell production, and thus simulate a disease process similar to human MDS. In our preliminary data, quantitative PCR and immunoblotting results confirm that human PKR is specifically expressed in spleen, thymus and bone marrow (BM) but not in liver, kidney, intestine, muscle and other tissues outside hematopoiesis in TgPKR mice. Aberrant activation of transcription factor STAT1 and MAPKinases ERK1/2 occurs in hematopoietic cells from the TgPKR mice. In addition, inhibitory cytokines including interferon gamma, CXCL9 and 10 are overexpressed. Initial characterization of BM cells from TgPKR mice indicates a higher level of uninduced/basal apoptosis in growth medium and increased apoptosis upon withdrawal of growth factors compared to cells from wild type (WT) littermates. Furthermore, BM cells from TgPKR mice display >20% reduced colonies (CFU-GM and CFU-GEMM) which contain 40% less cells than those from WT littermates. Significantly, histopathological analysis of BM and spleen from TgPKR mice reveals a ∼20% increase in bone marrow cellularity with a striking increase in dysplastic megakaryocytes that are either small without nuclear lobation or of normal size but with hypolobation or separated nuclear lobes. Taken together our results demonstrate that transgenic expression of hPKR induces an MDS-like condition in mice characterized by reduced BM cell survival, increased apoptosis and reduced proliferation and differentiation of BM progenitor/stem cells. Thus, increased PKR expression in patients may, at least in part, lead to a BM failure state similar to MDS. Thus, future anti-MDS therapies that inhibit PKR may be clinically useful. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1350. doi:1538-7445.AM2012-1350

Extensive synaptic and neuritic alterations in the neocortex and limbic system are characteristically found in Alzheimer's disease (AD). However, it is not known how early in the development of the disease these alterations occur. For the present study, we compared the synaptic and neuritic alterations among cases classified clinically and neuropathologically as early, mild and advanced AD. In early AD there was a 20% loss of synaptophysin-immunoreactive presynaptic terminals in the outer molecular layer of the hippocampal dentate gyrus (but not in the neocortex and entorhinal cortex), accompanied by increased amyloid precursor protein (APP) and Alz50 immunoreactivity in hippocampal and entorhinal cortex pyramidal neurons. These results suggest that abnormal neuronal expression of APP and cytoskeletal proteins in early stages might be involved in the mechanisms of synaptic pathology in AD.