Sting and p53 DNA repair pathways are compromised in Alzheimer's disease.

Alzheimer's disease (AD) is the most common cause of dementia. A common finding in AD is DNA damage. Double-strand DNA breaks (DSBs) are particularly hazardous to neurons because their post-mitotic state forces neurons to rely on error-prone and potentially mutagenic mechanisms to repair DNA breaks. However, it remains unclear whether DNA damage results from increased DNA damage or failure of DNA repair. Oligomerization of the tumor suppressor protein p53 is an essential part of DSB repair, and p53 phosphorylated on S15 is an indicator of DNA damage. We report that the monomer:dimer ratio of phosphorylated (S15) p53 is increased by 2.86-fold in temporal lobes of AD patients compared to age-matched controls, indicating that p53 oligomerization is compromised in AD. In vitro oxidation of p53 with 100 nM H2O2 produced a similar shift in the monomer:dimer ratio. A COMET test showed a higher level of DNA degradation in AD consistent with double-strand DNA damage or inhibition of repair. Protein carbonylation was also elevated (190% of control), indicating elevated oxidative stress in AD patients. Levels of the DNA repair support protein 14-3-3σ, γ-H2AX, a phosphorylated histone marking double strand DNA breaks, and phosphorylated ataxia telangiectasia mutated (ATM) protein were all increased. cGAS-STING-interferon signaling was impaired in AD and was accompanied by a depletion of STING protein from Golgi and a failure to elevate interferon despite the presence of DSBs. The results suggest that oxidation of p53 by ROS could inhibit the DDR and decrease its ability to orchestrate DSB repair by altering the oligomerization state of p53. The failure of immune-stimulated DNA repair may contribute to cell loss in AD and suggests new therapeutic targets for AD.

Modulation of proteasome activity by curcumin and didemethylcurcumin.

Modulation of proteasome function by pharmacological interventions and molecular biology tools is an active area of research in cancer biology and neurodegenerative diseases. Curcumin (diferuloylmethane) is a naturally occurring polyphenol that affects multiple signaling pathways. Curcumin shows anti-inflammatory, antioxidant, anti-angiogenic, or anti-apoptotic properties. Recent research suggests that the therapeutic efficacy of curcumin may be due to its activity as a potent inhibitor of the proteasome. Using in vitro cell culture and molecular docking methods, here we show that both curcumin and its synthetic polyphenolic derivative (didemethylcurcumin, CUIII) modulated proteasome activity in a biphasic manner. Curcumin and CUIII increased proteasome activity at nanomolar concentrations, but inhibited proteasome activity at micromolar concentrations. Curcumin was more effective than CUIII in increasing relative proteasome activity at nanomolar concentrations. Also, curcumin was more effective than CUIII in inhibiting relative proteasome activity at micromolar concentrations. Docking simulations of curcumin and didemethylcurcumin binding to the 20S proteasome catalytic subunit estimated Kd values of 0.0054 µM and 1.3167 µM, respectively. These values correlate well with the results of the effectiveness of modulation by curcumin compared to CUIII. The small size of CUIII allows it to dock to the narrow cavity of the active site, but the binding interaction is not strong compared to curcumin. These results indicate that curcumin and its didemethyl derivative can be used to modulate proteasome activity and suggest that curcumin and its didemethyl derivative may be useful in treating two different disease classes: neurodegeneration and cancer.Communicated by Ramaswamy H. Sarma.

Morphology of the nutrient artery and its foramen in relation to femoral bone perfusion rates of laying and non-laying hens.

If arteries penetrate bones through foramina, regional artery blood flow rates can be estimated from the foramen sizes. Femoral bone blood flow rates estimated from nutrient foramen sizes were previously not absolute, but only a relative blood flow index (Qi ), because the size relationship between the foramen and the occupying artery was unknown. The current study used vascular contrast and micro-computerized tomographic scanning to investigate femoral nutrient foramen and nutrient artery sizes in three groups of sub-adult chickens (non-laying hens, laying hens, and roosters) of similar ages. The results indicate that the cross-sectional area of the nutrient artery lumen occupies approximately 20.2 ± 4.1% of the foramen for femora with only one foramen. Artery lumen size is significantly correlated with foramen size. Vascular contrast imaging is capable of estimating blood flow rates through nutrient arteries, as blood flow rates estimated from artery lumen casts are similar to blood flow rates measured by infusion of fluorescent-labeled microspheres. Laying hens tend to have higher nutrient artery perfusion rates than non-laying hens, probably due to extra oxygen and calcium requirements for eggshell production, although the calculated blood flow difference was not statistically significant. Histological embedding and sectioning along with vascular contrast imaging reveal variable nutrient foramen morphology and nutrient artery location among femora with more than one nutrient foramen.

Regional femoral bone blood flow rates in laying and non-laying chickens estimated with fluorescent microspheres.

The metabolic rate of vertebrate bone tissue is related to bone growth, repair and homeostasis, which are all dependent on life stage. Bone metabolic rate is difficult to measure directly, but absolute blood flow rate () should reflect local tissue oxygen requirements. A recent 'foramen technique' has derived an index of blood flow rate () by measuring nutrient foramen sizes of long bones. is assumed to be proportional to ; however, the assumption has never been tested. This study used fluorescent microsphere infusion to measure femoral bone in anaesthetized non-laying hens, laying hens and roosters. Mean mass-specific cardiac output was 338±38 ml min-1 kg-1, and the two femora received 0.63±0.10% of this. Laying hens had higher wet bone mass-specific to femora (0.23±0.09 ml min-1 g-1) than the non-laying hens (0.12±0.06 ml min-1 g-1) and roosters (0.14±0.04 ml min-1 g-1), presumably associated with higher bone calcium mobilization during eggshell production. Estimated metabolic rate of femoral bone was 0.019 ml O2 min-1 g-1. Femoral increased significantly with body mass, but was not correlated with nutrient foramen radius (r), probably because of a narrow range in foramen radius. Over all 18 chickens, femoral shaft was 1.07±0.30 ml min-1 mm-1. Mean in chickens was significantly higher than predicted by an allometric relationship for adult cursorial bird species, possibly because the birds were still growing.

Bone foramen dimensions and blood flow calculation: best practices.

Some blood vessels enter bones through foramina, leaving the size of the foramen as a gauge for estimating the rate of blood flow and hence the metabolic rate of the supplied tissues. Foramen dimensions have been measured using varied methods in previous foramen studies, to relate regional blood flows with associated physiological processes. With the increasing interests in this 'foramen technique', standard methods with minimized measurement errors are therefore required. This study provides details of microphotographic and micro-computerized tomographic methods, and introduces a new alternative method, which uses impression material to measure foramen dimensions. The three methods are compared and the results indicate that all of them are capable of obtaining precise and accurate foramen dimension values, although they all have limitations. A microphotograph of the external opening is suggested to be the standard method because of its ease of use, but the alternative methods provide more detailed information on foramen shape. If the foramen is mainly occupied by one artery, blood flow rates can be calculated from foramen size and artery wall-lumen ratio, which is evaluated from the literature survey in this study. If veins or nerves also penetrate the foramen, a relative index of blood flow rate is nevertheless possible for comparative purposes.

Cerebral blood flow rates in recent great apes are greater than in Australopithecus species that had equal or larger brains.

Brain metabolic rate (MR) is linked mainly to the cost of synaptic activity, so may be a better correlate of cognitive ability than brain size alone. Among primates, the sizes of arterial foramina in recent and fossil skulls can be used to evaluate brain blood flow rate, which is proportional to brain MR. We use this approach to calculate flow rate in the internal carotid arteries (Q˙ICA), which supply most of the primate cerebrum. Q˙ICA is up to two times higher in recent gorillas, chimpanzees and orangutans compared with 3-million-year-old australopithecine human relatives, which had equal or larger brains. The scaling relationships between Q˙ICA and brain volume (Vbr) show exponents of 1.03 across 44 species of living haplorhine primates and 1.41 across 12 species of fossil hominins. Thus, the evolutionary trajectory for brain perfusion is much steeper among ancestral hominins than would be predicted from living primates. Between 4.4-million-year-old Ardipithecus and Homo sapiens, Vbr increased 4.7-fold, but Q˙ICA increased 9.3-fold, indicating an approximate doubling of metabolic intensity of brain tissue. By contrast, Q˙ICA is proportional to Vbr among haplorhine primates, suggesting a constant volume-specific brain MR.

Apolipoprotein E particle size is increased in Alzheimer's disease.

INTRODUCTION: Apolipoprotein E4 (apoE4) is the predominant risk factor for late-onset Alzheimer's disease (AD), but the question of which structural differences might explain its effect remains unclear. METHODS: We compared high-density lipoprotein-like apoE particles from 12 AD and 10 control patients using size-exclusion chromatography. RESULTS: ApoE particles from patients genotyped as ε4/ε4 were 2.2 ± 0.3 times as massive as particles from ε3/ε3 control subjects and 1.4 ± 0.1 times as massive as particles from ε3/ε3 AD patients. The increased particle size was not because of incorporation of amyloid ß or apoE proteolysis products. Particles from AD patients genotyped as ε3/ε3 were 1.59 ± 0.27 times as massive as ε3/ε3 control subjects. DISCUSSION: Increased particle size in AD is affected by A PO E genotype and by disease-related differences in assembly or stability. These differences suggest that lipoprotein assembly or stability in AD brain plays an important role in determining apoE4 pathogenicity.

Loss in PKC Epsilon Causes Downregulation of MnSOD and BDNF Expression in Neurons of Alzheimer's Disease Hippocampus.

Oxidative stress and amyloid-ß (Aß) oligomers have been implicated in Alzheimer's disease (AD). The growth and maintenance of neuronal networks are influenced by brain derived neurotrophic factor (BDNF) expression, which is promoted by protein kinase C epsilon (PKCɛ). We investigated the reciprocal interaction among oxidative stress, Aß, and PKCɛ levels and subsequent PKCɛ-dependent MnSOD and BDNF expression in hippocampal pyramidal neurons. Reduced levels of PKCɛ, MnSOD, and BDNF and an increased level of Aß were also found in hippocampal neurons from autopsy-confirmed AD patients. In cultured human primary hippocampal neurons, spherical aggregation of Aß (amylospheroids) decreased PKCɛ and MnSOD. Treatment with t-butyl hydroperoxide (TBHP) increased superoxide, the oxidative DNA/RNA damage marker, 8-OHG, and Aß levels, but reduced PKCɛ, MnSOD, BDNF, and cultured neuron density. These changes were reversed with the PKCɛ activators, bryostatin and DCPLA-ME. PKCɛ knockdown suppressed PKCɛ, MnSOD, and BDNF but increased Aß. In cultured neurons, the increase in reactive oxygen species (ROS) associated with reduced PKCɛ during neurodegeneration was inhibited by the SOD mimetic MnTMPyP and the ROS scavenger NAc, indicating that strong oxidative stress suppresses PKCɛ level. Reduction of PKCɛ and MnSOD was prevented with the PKCɛ activator bryostatin in 5-6-month-old Tg2576 AD transgenic mice. In conclusion, oxidative stress and Aß decrease PKCɛ expression. Reciprocally, a depression of PKCɛ reduces BDNF and MnSOD, resulting in oxidative stress. These changes can be prevented with the PKCɛ-specific activators.

The Initial Impact of Tele-Critical Care on the Surgical Services of a Community Military Hospital.

Introduction: Mortality is reduced in hospitals staffed with intensivists, however, many smaller military hospitals lack intensivist support. Naval Hospital Camp Pendleton (NHCP) is a Military Treatment Facility (MTF) that operates a 6-bed Intensive Care Unit (ICU) north of its referral center, Naval Medical Center San Diego (NMCSD). To address a gap in NHCP on-site intensivist coverage, a comprehensive Tele-Critical Care (TCC) support system was established between NHCP and NMCSD. To examine the initial impact of telemedicine on surgical ICU patients, we compare NHCP surgical ICU admissions before and after TCC implementation. Materials and methods: Patient care by remote intensivist was achieved utilizing video teleconferencing technology, and remote access to electronic medical records. Standardization was promoted by adopting protocols and mandatory intensivist involvement in all ICU admissions. Surgical ICU admissions prior to TCC implementation (pre-TCC) were compared to those following TCC implementation (post-TCC). Results: Of 828 ICU admissions, 21% were surgical. TCC provided coverage during 35% of the intervention period. Comparing pre-TCC and post-TCC periods, there was a significant increase in the percentage of surgical ICU admissions [15.3 % vs 24.6%, p = 0.01] and the average monthly APACHE II score [4.1vs 6.5, p = 0.03]. The total number of surgical admissions per month also increased [3.9 vs 6.3, p = 0.009]. No adverse outcomes were identified. Conclusion: Implementation of TCC was associated with an increase in the scope and complexity of surgical admissions with no adverse outcomes. Surgeons were able to safely expand the surgical services offered requiring perioperative ICU care to patients who previously may have been transferred. Caring for these types of patients not only maintains the operational readiness of deployable caregivers but patient experience is also enhanced by minimizing transfers away from family. Further exploration of TCC on surgical case volume and complexity is warranted.

Protein kinase C activator bryostatin-1 modulates proteasome function.

Proteasome activity in ubiquitin-proteasome pathway plays a pivotal role in degradation and clearance of aggregated, oxidized, damaged, and misfolded unwanted proteins to control protein homeostasis or proteostasis. Proteasome activity decreases with cellular senescence, aging, and age-related diseases. Therefore, enhancement of impaired proteasome function by molecular biological and/or pharmacological intervention is an active area of research. Bryostatin-1, a naturally occurring macrocyclic lactone, activates PKC isozymes (specifically, -α and -ϵ) at sub-nanomolar concentrations, but downregulates at higher concentrations. Here, we present bryostatin-1 increased chymotrypsin-like proteasome activity of 20S assembly at sub-nanomolar to nanomolar concentrations (0.3-30 nM). However, proteasome activity decreased at a micromolar concentration of bryostatin-1 (AG08044 cultured skin: P < 0.005; differentiated SH-SY5Y cells: P < 0.02). Modulation of proteasome function by bryostatin-1 was studied in six dermal fibroblast primary cell lines developed both from freshly taken biopsies from healthy donors (n = 2) and obtained from well-characterized cell repositories (n = 4; without any diseases). Bryostatin-1 enhanced proteasome activity in cultured skin fibroblasts obtained from banked and freshly isolated skin fibroblasts from skin biopsies at the sub-nanomolar concentration (P < 0.015). Modulation of proteasome function by bryostatin-1 was confirmed in neuron-like differentiated SH-SY5Y cells. Direct additions of bryostatin-1 into cell lysates prepared from neuron-like differentiated SH-SY5Y, Jurkat cells, and cultured skin fibroblasts were unable to increase proteasome activity indicating that bryostatin-1 can only modulate proteasome activity when added to live cell culture systems. Standard PKC inhibitors blocked bryostatin-1 induced proteasome activity modulation suggesting that enhancement of proteasome activity was mediated by PKC modulation.

Femoral bone perfusion through the nutrient foramen during growth and locomotor development of western grey kangaroos (Macropus fuliginosus).

The nutrient artery passes through the nutrient foramen on the shaft of the femur and supplies more than half of the total blood flow to the bone. Assuming that the size of the nutrient foramen correlates with the size of the nutrient artery, an index of blood flow rate (Qi) can be calculated from nutrient foramen dimensions. Interspecific Qi is proportional to locomotor activity levels in adult mammals, birds and reptiles. However, no studies have yet estimated intraspecific Qi to test for the effects of growth and locomotor development on bone blood flow requirements. In this study, we used micro-CT and medical CT scanning to measure femoral dimensions and foramen radius to calculate femoral Qi during the in-pouch and post-pouch life stages of western grey kangaroos (Macropus fuliginosus) weighing 5.7 g to 70.5 kg and representing a 12,350-fold range in body mass. A biphasic scaling relationship between Qi and body mass was observed (breakpoint at ca. 1-5 kg body mass right before permanent pouch exit), with a steep exponent of 0.96±0.09 (95% CI) during the in-pouch life stage and a statistically independent exponent of -0.59±0.90 during the post-pouch life stage. In-pouch joeys showed Qi values that were 50-100 times higher than those of adult diprotodont marsupials of the same body mass, but gradually converged with them as post-pouch adults. Bone modelling during growth appears to be the main determinant of femoral bone blood flow during in-pouch development, whereas bone remodelling for micro-fracture repair due to locomotion gradually becomes the main determinant when kangaroos leave the pouch and become more active.

ApoE isoforms differentially regulates cleavage and secretion of BDNF.

Apolipoprotein E4 (ApoE4) is a major genetic risk factor for sporadic or late onset Alzheimer's disease (AD). Brain-derived neurotrophic factor (BDNF) is decreased by 3 to 4-fold in the brains of AD patients at autopsy. ApoE4 mice also have reduced BDNF levels. However, there have been no reports relating the different ApoE isoforms or AD to differential regulation of BDNF. Here we report that in the hippocampal regions of AD patients both prepro-BDNF and pro-BDNF expression showed a 40 and 60% decrease respectively compared to that expression in the hippocampi of age-matched control patients. We further report that ApoE isoforms differentially regulate maturation and secretion of BDNF from primary human astrocytes. After 24 h, ApoE3 treated astrocytes secreted 1.75- fold higher pro-BDNF than ApoE2-treated astrocytes, and ApoE2-treated astrocytes secreted 3-fold more mature-BDNF (m-BDNF) than ApoE3-treated astrocytes. In contrast, ApoE4-treated cells secreted negligible amounts of m-BDNF or pro-BDNF. ApoE2 increased the level of intracellular pre-pro BDNF by 19.04 ± 6.68%, while ApoE4 reduced the pre-pro BDNF by 21.61 ± 5.9% compared to untreated cells. Similar results were also seen in ApoE2, ApoE3 or ApoE4 treated cells at 4 h. Together, these results indicate that an ApoE2 or ApoE3 mediated positive regulation of BDNF may be protective while ApoE4 related defects in BDNF processing could lead to AD pathophysiology. These interactions of the ApoE isoforms with BDNF may help explain the increased risk of AD associated with the ApoE4 isoform.

Bryostatin Effects on Cognitive Function and PKCɛ in Alzheimer's Disease Phase IIa and Expanded Access Trials.

Bryostatin 1, a potent activator of protein kinase C epsilon (PKCɛ), has been shown to reverse synaptic loss and facilitate synaptic maturation in animal models of Alzheimer's disease (AD), Fragile X, stroke, and other neurological disorders. In a single-dose (25 µg/m2) randomized double-blind Phase IIa clinical trial, bryostatin levels reached a maximum at 1-2 h after the start of infusion. In close parallel with peak blood levels of bryostatin, an increase of PBMC PKCɛ was measured (p = 0.0185) within 1 h from the onset of infusion. Of 9 patients with a clinical diagnosis of AD, of which 6 received drug and 3 received vehicle within a double-blind protocol, bryostatin increased the Mini-Mental State Examination (MMSE) score by +1.83±0.70 unit at 3 h versus -1.00±1.53 unit for placebo. Bryostatin was well tolerated in these AD patients and no drug-related adverse events were reported. The 25 µg/m2 administered dose was based on prior clinical experience with three Expanded Access advanced AD patients treated with bryostatin, in which return of major functions such as swallowing, vocalization, and word recognition were noted. In one Expanded Access patient trial, elevated PKCɛ levels closely tracked cognitive benefits in the first 24 weeks as measured by MMSE and ADCS-ADL psychometrics. Pre-clinical mouse studies showed effective activation of PKCɛ and increased levels of BDNF and PSD-95. Together, these Phase IIa, Expanded Access, and pre-clinical results provide initial encouragement for bryostatin 1 as a potential treatment for AD.

Protein Kinase Cϵ (PKCϵ) Promotes Synaptogenesis through Membrane Accumulation of the Postsynaptic Density Protein PSD-95.

Protein kinase Cϵ (PKCϵ) promotes synaptic maturation and synaptogenesis via activation of synaptic growth factors such as BDNF, NGF, and IGF. However, many of the detailed mechanisms by which PKCϵ induces synaptogenesis are not fully understood. Accumulation of PSD-95 to the postsynaptic density (PSD) is known to lead to synaptic maturation and strengthening of excitatory synapses. Here we investigated the relationship between PKCϵ and PSD-95. We show that the PKCϵ activators dicyclopropanated linoleic acid methyl ester and bryostatin 1 induce phosphorylation of PSD-95 at the serine 295 residue, increase the levels of PSD-95, and enhance its membrane localization. Elimination of the serine 295 residue in PSD-95 abolished PKCϵ-induced membrane accumulation. Knockdown of either PKCϵ or JNK1 prevented PKCϵ activator-mediated membrane accumulation of PSD-95. PKCϵ directly phosphorylated PSD-95 and JNK1 in vitro Inhibiting PKCϵ, JNK, or calcium/calmodulin-dependent kinase II activity prevented the effects of PKCϵ activators on PSD-95 phosphorylation. Increase in membrane accumulation of PKCϵ and phosphorylated PSD-95 (p-PSD-95(S295)) coincided with an increased number of synapses and increased amplitudes of excitatory post-synaptic potentials (EPSPs) in adult rat hippocampal slices. Knockdown of PKCϵ also reduced the synthesis of PSD-95 and the presynaptic protein synaptophysin by 30 and 44%, respectively. Prolonged activation of PKCϵ increased synapse number by 2-fold, increased presynaptic vesicle density, and greatly increased PSD-95 clustering. These results indicate that PKCϵ promotes synaptogenesis by activating PSD-95 phosphorylation directly through JNK1 and calcium/calmodulin-dependent kinase II and also by inducing expression of PSD-95 and synaptophysin.

ApoE4 and Aß Oligomers Reduce BDNF Expression via HDAC Nuclear Translocation.

Apolipoprotein E4 (ApoE4) is a major genetic risk factor for several neurodegenerative disorders, including Alzheimer's disease (AD). Epigenetic dysregulation, including aberrations in histone acetylation, is also associated with AD. We show here for the first time that ApoE4 increases nuclear translocation of histone deacetylases (HDACs) in human neurons, thereby reducing BDNF expression, whereas ApoE3 increases histone 3 acetylation and upregulates BDNF expression. Amyloid-ß (Aß) oligomers, which have been implicated in AD, caused effects similar to ApoE4. Blocking low-density lipoprotein receptor-related protein 1 (LRP-1) receptor with receptor-associated protein (RAP) or LRP-1 siRNA abolished the ApoE effects. ApoE3 also induced expression of protein kinase C ε (PKCε) and PKCε retained HDACs in the cytosol. PKCε activation and ApoE3 supplementation prevented ApoE4-mediated BDNF downregulation. PKCε activation also reversed Aß oligomer- and ApoE4-induced nuclear import of HDACs, preventing the loss in BDNF. ApoE4 induced HDAC6-BDNF promoter IV binding, which reduced BDNF exon IV expression. Nuclear HDAC4 and HDAC6 were more abundant in the hippocampus of ApoE4 transgenic mice than in ApoE3 transgenic mice or wild-type controls. Nuclear translocation of HDA6 was also elevated in the hippocampus of AD patients compared with age-matched controls. These results provide new insight into the cause of synaptic loss that is the most important pathologic correlate of cognitive deficits in AD.

Towards universal therapeutics for memory disorders.

Evidence is accumulating that many memory disorders, including those due to neurodegenerative diseases, traumatic brain injury (TBI), vascular disease, or abnormal brain development, share common features of memory-related pathology. Structural and functional deficits of synapses are at the core of the underlying pathophysiology, constituting a critical point of convergence in memory disorders. Memory therapeutics that target synaptic loss and dysfunction - that is, to slow, halt, or reverse progression of the disorders at the level of synapses, via synaptogenic molecular cascades such as those of protein kinase C (PKC) and brain-derived neurotrophic factor (BDNF) - possess universal therapeutic value for many forms of memory disorder. They may be useful either as standalone interventions for patients with memory disorders or as adjuncts to drugs that target the underlying pathology.

PKCε deficits in Alzheimer's disease brains and skin fibroblasts.

In Alzheimer's disease (AD) transgenic mice, activation of synaptogenic protein kinase C ε (PKCε) was found to prevent synaptotoxic amyloid-ß (Aß)-oligomer elevation, PKCε deficits, early synaptic loss, cognitive deficits, and amyloid plaque formation. In humans, to study the role of PKCε in the pathophysiology of AD and to evaluate its possible use as an early AD-biomarker, we examined PKCε and Aß in the brains of autopsy-confirmed AD patients (n = 20) and age-matched controls (AC, n = 19), and in skin fibroblast samples from AD (n = 14), non-AD dementia patients (n = 14), and AC (n = 22). Intraneuronal Aß levels were measured immunohistochemically (using an Aß-specific antibody) in hippocampal pyramidal cells of human autopsy brains. PKCε was significantly lower in the hippocampus and temporal pole areas of AD brains, whereas Aß levels were significantly higher. The ratio of PKCε to Aß in individual CA1 pyramidal cells was markedly lower in the autopsy AD brains versus controls. PKCε was inversely correlated with Aß levels in controls, whereas in AD patients, PKCε showed no significant correlation with Aß. In autopsy brains, PKCε decreased as the Braak score increased. Skin fibroblast samples from AD patients also demonstrated a deficit in PKCε compared to controls and an AD-specific change in the Aß-oligomer effects on PKCε. Together, these data demonstrate that the relationship between Aß levels and PKCε is markedly altered in AD patients' brains and skin fibroblasts, reflecting a loss of protective effect of PKCε against toxic Aß accumulation. These changes of PKCε levels in human skin fibroblasts may provide an accurate, non-invasive peripheral AD biomarker.

Molecular regulation of synaptogenesis during associative learning and memory.

Synaptogenesis plays a central role in associative learning and memory. The biochemical pathways that underlie synaptogenesis are complex and incompletely understood. Nevertheless, research has so far identified three conceptually distinct routes to synaptogenesis: cell-cell contact mediated by adhesion proteins, cell-cell biochemical signaling from astrocytes and other cells, and neuronal signaling through classical ion channels and cell surface receptors. The cell adhesion pathways provide the physical substrate to the new synaptic connection, while cell-cell signaling may provide a global or regional signal, and the activity-dependent pathways provide the neuronal specificity that is required for the new synapses to produce functional neuronal networks capable of storing associative memories. These three aspects of synaptogenesis require activation of a variety of interacting biochemical pathways that converge on the actin cytoskeleton and strengthen the synapse in an information-dependent manner. This article is part of a Special Issue titled SI: Brain and Memory.

Adduct formation in liquid chromatography-triple quadrupole mass spectrometric measurement of bryostatin 1.

Bryostatin 1, a potential anti-Alzheimer drug, is effective at subnanomolar concentrations. Measurement is complicated by the formation of low m/z degradation products and the formation of adducts with various cations, which make accurate quantitation difficult. Adduct formation caused the sample matrix or mobile phase to partition bryostatin 1 into products of different mass. Degradation of the 927 [M+Na](+) ion to a 869m/z product was strongly influenced by ionization conditions. We validated a bryostatin 1 assay in biological tissues using capillary column HPLC with nanospray ionization (NSI) in a triple-quadrupole mass spectrometer in selected reaction monitoring (SRM) mode. Adduct formation was controlled by adding 1mM acetic acid and 0.1mM sodium acetate to the HPLC buffer, maximizing the formation of the [M+Na](+) ion. Efficient removal of contaminating cholesterol from the sample during solvent extraction was also critical. The increased sensitivity provided by NSI and capillary-bore columns and the elimination of signal partitioning due to adduct formation and degradation in the ionization source enabled a detection limit of 1×10(-18)mol of bryostatin 1 and a LLOQ of 3×10(-18)mol from 1µl of sample. Bryostatin 1 at low pmol/l concentrations enabled measurement in brain and other tissues without the use of radioactive labels. Despite bryostatin 1's high molecular weight, considerable brain access was observed, with peak brain concentrations exceeding 8% of the peak blood plasma concentrations. Bryostatin 1 readily crosses the blood-brain barrier, reaching peak concentrations of 0.2nM, and specifically activates and translocates brain PKCɛ.