The karyometric signature is altered in fallopian tubes with serous tubal intraepithelial carcinoma.

OBJECTIVE: Recent evidence suggests that the fimbriated end of the fallopian tube harbors the precursor cells for many high-grade ovarian cancers, opening the door for development of better screening methods that directly assess the fallopian tube in women at risk for malignancy. Previously we have shown that the karyometric signature is abnormal in the fallopian tube epithelium in women at hereditary risk of ovarian cancer. In this study, we sought to determine whether the karyometric signature in serous tubal intraepithelial carcinoma (STIC) is significantly different from normal, and whether an abnormal karyometric signature can be detected in histologically normal tubal epithelial cells adjacent to STIC lesions. METHODS: The karyometric signature was measured in epithelial cells from the proximal and fimbriated portion of the fallopian tube in fallopian tube specimens removed from women at: 1) average risk for ovarian cancer undergoing surgery for benign gynecologic indications (n = 37), 2) hereditary risk of ovarian cancer (germline BRCA alterations) undergoing risk-reducing surgery (n = 44), and 3) diagnosed with fimbrial STICs (n = 17). RESULTS: The karyometric signature in tubes with fimbrial STICs differed from that of tubes with benign histology. The degree of karyometric alteration increased with increasing proximity to fimbrial STICs, ranging from moderate in the proximal portion of the tube, to greatest in both normal appearing fimbrial cells near STICs as well as in fimbrial STIC lesions. CONCLUSION: These data demonstrate an abnormal karyometric signature in STICs that may extend beyond the STIC, potentially providing an opportunity for early detection of fallopian tube neoplasia.

Determination of multiclass pesticides in river sediments via matrix solid-phase dispersion extraction and gas chromatography-tandem mass spectrometry.

A fast and environment-friendly analytical method was implemented to determine multiclass pesticides in river sediments. Twenty-three pesticides-organochlorine pesticides, organophosphorus pesticides, and triazines-were extracted via matrix solid-phase dispersion (MSPD) and analyzed by gas chromatography-tandem mass spectrometry (GC-MS/MS). Florisil demonstrated excellent analytes uptake capability as the extractant phase, with suitable selectivity for treating complex sediment samples. Under defined extraction conditions, the MSPD-GC-MS/MS method demonstrated robustness in the n inter-day analysis of sediments from different sources, providing limit of quantifications (LOQs) between 5 and 15 ng/g, linear responses in the range between LOQs and 150 ng/g, extraction recoveries of 71%-106%, and precision, assessed as relative standard deviation below 20%. The MSPD significantly reduced samples and solvents' consumption, providing critical environmental gains compared to traditional extraction methods like Soxhlet. Finally, the method was applied to analyze sediment samples from three different collection areas of the Subachoque River (Cundinamarca, Colombia), demonstrating a fast, efficient, confident, and profitable analytical tool for pollution control and monitoring in environmental samples. The method allowed us to determine the current use in Colombia of banned pesticides under the 2001 Stockholm Convention.

Progestin Significantly Inhibits Carcinogenesis in the Mogp-TAg Transgenic Mouse Model of Fallopian Tube Cancer.

Recent studies suggest that the fallopian tube epithelium (FTE) harbors the precursor for high-grade ovarian cancer, creating opportunities for targeting the FTE for ovarian cancer prevention. Preclinical evidence supports progestins as ovarian cancer preventives, but the effect of progestins on the FTE is not well characterized. The murine oviduct-specific glycoprotein promotor-driven simian virus 40 large T-Antigen (mogp-TAg) transgenic mouse model develops neoplastic lesions in the fallopian tube in a manner similar to that described in human fallopian tube and ovarian cancers. In this study, we investigated the inhibitory effects of the progestin depo-medroxyprogesterone acetate (DMPA) on fallopian tube carcinogenesis following treatment for 3 and 7 weeks in 5-week-old mogp-TAg mice. Overall, compared with vehicle-treated mice, the fallopian tube of DMPA-treated mice was significantly smaller (P < 0.0005), accumulated fewer p53-positive cells, had normal distribution of ciliated cells, less nuclear pleomorphism and epithelial tufting, and had a significantly lower proliferative index (P = 0.001). Accumulation of p53 signatures and serous tubal intraepithelial carcinomas (STIC) in the fallopian tube was significantly reduced in the DMPA (P < 0.0005) treatment group. Moreover, the fallopian tube of the DMPA-treated mice developed significantly less adenocarcinoma compared with vehicle (P < 0.005) at both treatment time points. DMPA treatment significantly induced cleaved caspase-3 (P < 0.0005) in the FTE compared with vehicle suggesting that apoptosis is involved in DMPA-related clearance of abnormal cells from the fallopian tube. These data demonstrate that DMPA targets early events in fallopian tube carcinogenesis by clearing genetically damaged cells, leading to marked reduction in adenocarcinoma, supporting progestins as chemopreventive agents for fallopian tube and ovarian cancers. PREVENTION RELEVANCE: The fallopian tube is thought to harbor the cell of origin for most ovarian cancers. We show in a mouse model of fallopian tube cancer that progestin eradicates the earliest known precancerous lesions and markedly inhibits fallopian tube carcinogenesis, adding to growing preclinical evidence supporting progestins as potent ovarian cancer chemopreventive agents.

Sub-millimeter endoscope demonstrates feasibility of in vivo reflectance imaging, fluorescence imaging, and cell collection in the fallopian tubes.

SIGNIFICANCE: Most cases of high-grade serous ovarian carcinoma originate as serous tubal intraepithelial carcinoma (STIC) lesions in the fallopian tube epithelium (FTE), enabling early endoscopic detection. AIM: The cell-acquiring fallopian endoscope (CAFE) was built to meet requirements for locating potentially pathological tissue indicated by an alteration in autofluorescence or presence of a targeted fluorophore. A channel was included for directed scrape biopsy of cells from regions of interest. APPROACH: Imaging resolution and fluorescence sensitivity were measured using a standard resolution target and fluorescence standards, respectively. A prototype was tested in ex vivo tissue, and collected cells were counted and processed. RESULTS: Measured imaging resolution was 88 µm at a 5-mm distance, and full field of view was ∼45 deg in air. Reflectance and fluorescence images in ex vivo porcine reproductive tracts were captured, and fit through human tracts was verified. Hemocytometry counts showed that on the order of 105 cells per scrape biopsy could be collected from ex vivo porcine tissue. CONCLUSIONS: All requirements for viewing STIC in the FTE were met, and collected cell counts exceeded input requirements for relevant analyses. Our benchtop findings suggest the potential utility of the CAFE device for in vivo imaging and cell collection in future clinical trials.

Integrative Transcriptome Analyses of the Human Fallopian Tube: Fimbria and Ampulla-Site of Origin of Serous Carcinoma of the Ovary.

Epithelial ovarian cancer represents a group of heterogeneous diseases with high grade serous cancer (HGSC) representing the most common histotype. Molecular profiles of precancerous lesions found in the fallopian tube have implicated this tissue as the presumptive site of origin of HGSC. Precancerous lesions are primarily found in the distal fallopian tube (fimbria), near the ovary relative to the proximal tissue (ampulla), nearer to the uterus. The proximity of the fimbria to the ovary and the link between ovulation, through follicular fluid release, and ovarian cancer risk led us to examine transcriptional responses of fallopian tube epithelia (FTE) at the different anatomical sites of the human fallopian tube. Gene expression profiles of matched FTE from the fimbria and from premenopausal women resulted in differentially expressed genes (DEGs): CYYR1, SALL1, FOXP2, TAAR1, AKR1C2/C3/C4, NMBR, ME1 and GSTA2. These genes are part of the antioxidant, stem and inflammation pathways. Comparisons between the luteal phase (post-ovulation) to the follicular phase (pre-ovulation) demonstrated greater differences in DEGs than a comparison between fimbria and fallopian tube anatomical differences alone. This data suggests that cyclical transcriptional changes experienced in pre-menopause are inherent physiological triggers that expose the FTE in the fimbria to cytotoxic stressors. These cyclical exposures induce transcriptional changes reflective of genotoxic and cytotoxic damage to the FTE in the fimbria which are closely related to transcriptional and genomic alterations observed in ovarian cancer.

Association between C-reactive protein and radiotherapy-related pain in a tri-racial/ethnic population of breast cancer patients: a prospective cohort study.

BACKGROUND: Post-surgery adjuvant radiotherapy (RT) significantly improves clinical outcomes in breast cancer patients; however, some patients develop cancer or treatment-related pain that negatively impacts quality of life. This study examined an inflammatory biomarker, C-reactive protein (CRP), in RT-related pain in breast cancer. METHODS: During 2008 and 2014, breast cancer patients who underwent RT were prospectively evaluated for pre- and post-RT pain. Pre- and post-RT plasma CRP levels were measured using a highly sensitive CRP ELISA kit. Pain score was assessed as the mean of four pain severity items (i.e., pain at its worst, least, average, and now) from the Brief Pain Inventory. Pain scores of 4-10 were classified as clinically relevant pain. Multivariable logistic regression analyses were applied to ascertain the associations between CRP and RT-related pain. RESULTS: In 366 breast cancer patients (235 Hispanic whites, 73 black/African Americans, and 58 non-Hispanic whites), 17% and 30% of patients reported pre- and post-RT pain, while 23% of patients had RT-related pain. Both pre- and post-RT pain scores differed significantly by race/ethnicity. In multivariable logistic regression analysis, RT-related pain was significantly associated with elevated pre-RT CRP (≥ 10 mg/L) alone (odds ratio (OR) = 2.44; 95% confidence interval (CI) = 1.02, 5.85); or combined with obesity (OR = 4.73; 95% CI = 1.41, 15.81) after adjustment for age and race/ethnicity. CONCLUSIONS: This is the first pilot study of CRP in RT-related pain, particularly in obese breast cancer patients. Future larger studies are warranted to validate our findings and help guide RT decision-making processes and targeted interventions.

Mutational re-modeling of di-aspartyl intramembrane proteases: uncoupling physiologically-relevant activities from those associated with Alzheimer's disease.

The intramembrane proteolytic activities of presenilins (PSEN1/PS1 and PSEN2/PS2) underlie production of ß-amyloid, the key process in Alzheimer's disease (AD). Dysregulation of presenilin-mediated signaling is linked to cancers. Inhibition of the γ-cleavage activities of PSENs that produce Aß, but not the ε-like cleavage activity that release physiologically essential transcription activators, is a potential approach for the development of rational therapies for AD. In order to identify whether different activities of PSEN1 can be dissociated, we designed multiple mutations in the evolutionary conserved sites of PSEN1. We tested them in vitro and in vivo assays and compared their activities with mutant isoforms of presenilin-related intramembrane di-aspartyl protease (IMPAS1 (IMP1)/signal peptide peptidase (SPP)). PSEN1 auto-cleavage was more resistant to the mutation remodeling than the ε-like proteolysis. PSEN1 with a G382A or a P433A mutation in evolutionary invariant sites retains functionally important APP ε- and Notch S3- cleavage activities, but G382A inhibits APP γ-cleavage and Aß production and a P433A elevates Aß. The G382A variant cannot restore the normal cellular ER Ca2+ leak in PSEN1/PSEN2 double knockout cells, but efficiently rescues the loss-of-function (Egl) phenotype of presenilin in C. elegans. We found that, unlike in PSEN1 knockout cells, endoplasmic reticulum (ER) Ca2+ leak is not changed in the absence of IMP1/SPP. IMP1/SPP with the analogous mutations retained efficiency in cleavage of transmembrane substrates and rescued the lethality of Ce-imp-2 knockouts. In summary, our data show that mutations near the active catalytic sites of intramembrane di-aspartyl proteases have different consequences on proteolytic and signaling functions.

Effect of actionable somatic mutations on racial/ethnic disparities in head and neck cancer prognosis.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide and minorities have the worst survival. However, the molecular mechanisms underlying survival disparities have not been elucidated. METHODS: In a retrospective study, we assessed association between HNSCC early death (<2 years) and 208 somatic mutations of 10 cancer-related genes in 214 patients: 98 non-Hispanic whites (46%), 72 Hispanic whites (34%), and 44 African Americans (20%). RESULTS: Hispanic whites and African Americans had significantly higher mutation rates for EGFR, HRAS, KRAS, and TP53. HNSCC early death was significantly associated with 3+ mutations (odds ratio [OR] = 2.78, 95% confidence interval [CI] = 1.16, 6.69), NOTCH1 mutations in non-Hispanic whites (OR = 5.51; 95% CI = 1.22-24.83) and TP53 mutations in Hispanic whites (OR = 3.84; 95% CI = 1.08-13.68) in multivariable analysis adjusted for age, sex, tumor site, and tumor stage. CONCLUSION: We have provided the proof-of-principal data to link racial/ethnic-specific somatic mutations and HNSCC prognosis and pave the way for precision medicine to overcome HNSCC survival disparities. © 2016 Wiley Periodicals, Inc. Head Neck 38:1234-1241, 2016.

Prospective evaluation of radiation-induced skin toxicity in a race/ethnically diverse breast cancer population.

We evaluated predictors of radiation-induced skin toxicity in a prospective study of a tri-racial/ethnic breast cancer population. We evaluated patient demographics, tumor characteristics, and treatment variables in the first 392 patients in a prospective study assessing radiation-induced skin toxicity. Logistic regression analyses were conducted to evaluate potential predictors of skin toxicity. The study consists of 59 non-Hispanic whites (NHW; 15%), 241 Hispanic Whites (HW; 62%), 79 black or African Americans (AA; 20%), and 13 others (3%). Overall, 48% developed grade 0-1 skin toxicity, 49.8% grade 2, and 2.2% grade 3 by the National Cancer Institute's Common Toxicity Criteria for Adverse Events (CTCAE) scale. Twenty-one percent developed moist desquamation. In multivariate analysis, higher body mass index (BMI; OR = 2.09; 95%CI = 1.15, 3.82), higher disease stage (OR = 1.82; 95%CI = 1.06, 3.11), ER-positive/PR-negative status (OR = 2.74; 95%CI = 1.26, 5.98), and conventionally fractionated regimens (OR = 3.25; 95%CI = 1.76, 6.01) were significantly associated with higher skin toxicity grade after adjustment for age, race, ethnicity, ER status, and breast volume. B MI specifically predicted for moist desquamation, but not degree of erythema. In this racially and ethnically diverse cohort of breast cancer patients receiving radiation to the intact breast, risk factors including BMI, disease stage, and conventionally fractionated radiation predicted for higher skin toxicity grade, whereas age, race, ethnicity, and breast volume did not. BMI specifically predicted for moist desquamation, suggesting that preventive measures to address this particular outcome should be investigated.

Characterization of risk factors for adjuvant radiotherapy-associated pain in a tri-racial/ethnic breast cancer population.

Pain related to cancer or treatment is a critical quality of life issue for breast cancer survivors. In a prospective study of 375 patients with breast cancer (enrolled during 2008-2014), we characterized the risk factors for adjuvant radiotherapy (RT)-associated pain. Pain score was assessed at pre-RT and post-RT as the mean of 4 pain severity items (ie, pain at its worst, least, average, and now) from the Brief Pain Inventory with 11-point numeric rating scale (0-10). Pain scores of 4 to 10 were considered clinically relevant pain. The study consists of 58 non-Hispanic whites (15%), 78 black or African Americans (AA; 21%), and 239 Hispanic whites (HW; 64%). Overall, the prevalence of pre-RT, post-RT, and RT-associated clinically relevant pain was 16%, 31% and 20%, respectively. In univariate analysis, AA and HW had significantly higher pre-RT and post-RT pain than non-Hispanic whites. In multivariable logistic regression analysis, pre-RT pain was significantly associated with HW and obesity; post-RT pain was significantly associated with AA, HW, younger age, ≥2 comorbid conditions, above-median hotspot volume receiving >105% prescribed dose, and pre-RT pain score ≥4. Radiotherapy-associated pain was significantly associated with AA (odds ratio [OR] = 3.27; 95% confidence interval [CI] = 1.09-9.82), younger age (OR = 2.44, 95% CI = 1.24-4.79), and 2 or ≥3 comorbid conditions (OR = 3.06, 95% CI = 1.32-7.08; OR = 4.61, 95% CI = 1.49-14.25, respectively). These risk factors may help to guide RT decision-making process, such as hypofractionated RT schedule. Furthermore, effective pain management strategies are needed to improve quality of life in patients with breast cancer with clinically relevant pain.

Mutagenesis mapping of the presenilin 1 calcium leak conductance pore.

Missense mutations in presenilin 1 (PS1) and presenilin 2 (PS2) proteins are a major cause of familial Alzheimer disease. Presenilins are proteins with nine transmembrane (TM) domains that function as catalytic subunits of the γ-secretase complex responsible for the cleavage of the amyloid precursor protein and other type I transmembrane proteins. The water-filled cavity within presenilin is necessary to mediate the intramembrane proteolysis reaction. Consistent with this idea, cysteine-scanning mutagenesis and NMR studies revealed a number of water-accessible residues within TM7 and TM9 of mouse PS1. In addition to γ-secretase function, presenilins also demonstrate a low conductance endoplasmic reticulum Ca(2+) leak function, and many familial Alzheimer disease presenilin mutations impair this function. To map the potential Ca(2+) conductance pore in PS1, we systematically evaluated endoplasmic reticulum Ca(2+) leak activity supported by a series of cysteine point mutants in TM6, TM7, and TM9 of mouse PS1. The results indicate that TM7 and TM9, but not TM6, could play an important role in forming the conductance pore of PS1. These results are consistent with previous cysteine-scanning mutagenesis and NMR analyses of PS1 and provide further support for our hypothesis that the hydrophilic catalytic cavity of presenilins may also constitute a Ca(2+) conductance pore.

Familial Alzheimer's disease mutations in presenilins: effects on endoplasmic reticulum calcium homeostasis and correlation with clinical phenotypes.

Mutations in presenilins 1 and 2 (PS1 and PS2) are responsible for approximately 40% of all early onset familial Alzheimer's disease (FAD) monogenic cases. Presenilins (PSs) function as the catalytic subunit of γ-secretase and support cleavage of the amyloid-ß protein precursor (AßPP). We previously discovered that PSs also function as passive endoplasmic reticulum (ER) calcium (Ca2+) leak channels and that most FAD mutations in PSs affected their ER Ca2+ leak function. To further validate the relevance of our findings to human disease, we here performed Ca2+ imaging experiments with lymphoblasts established from FAD patients. We discovered that most FAD mutations in PSs disrupted ER Ca2+ leak function and resulted in increased ER Ca2+ pool in human lymphoblasts. However, we found that a subset of PS1 FAD mutants supported ER Ca2+ leak activity, as ER Ca2+ pool was unaffected in lymphoblasts. Most of the "functional" mutations for ER Ca2+ leak were clustered in the exon 8-9 area of PSEN1 gene and segregated with the cotton wool plaques and spastic paraparesis clinical phenotype occasionally observed in PS1 FAD patients. Our findings with the "functional" and "non-functional" PS1 FAD mutants were confirmed in Ca2+ rescue experiments with PS double-knockout mouse embryonic fibroblasts. Based on the combined effects of the PS1 FAD mutations on ER Ca2+ leak and γ-secretase activities we propose a model that explains the heterogeneity observed in FAD. The proposed model has implications for understanding the pathogenesis of both familial and sporadic AD.

Deranged calcium signaling and neurodegeneration in spinocerebellar ataxia type 2.

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominantly inherited, neurodegenerative disease caused by an expansion of polyglutamine tracts in the cytosolic protein ataxin-2 (Atx2). Cerebellar Purkinje cells (PCs) are predominantly affected in SCA2. The cause of PC degeneration in SCA2 is unknown. Here we demonstrate that mutant Atx2-58Q, but not wild-type (WT) Atx2-22Q, specifically associates with the cytosolic C-terminal region of type 1 inositol 1,4,5-trisphosphate receptor (InsP(3)R1), an intracellular calcium (Ca(2+)) release channel. Association with Atx2-58Q increased the sensitivity of InsP(3)R1 to activation by InsP(3) in planar lipid bilayer reconstitution experiments. To validate physiological significance of these findings, we performed a series of experiments with an SCA2-58Q transgenic mouse model that expresses human full-length Atx2-58Q protein under the control of a PC-specific promoter. In Ca(2+) imaging experiments, we demonstrated that stimulation with 3,5-dihydroxyphenylglycine (DHPG) resulted in higher Ca(2+) responses in 58Q PC cultures than in WT PC cultures. DHPG-induced Ca(2+) responses in 58Q PC cultures were blocked by the addition of ryanodine, an inhibitor of the ryanodine receptor (RyanR). We further demonstrated that application of glutamate induced more pronounced cell death in 58Q PC cultures than in WT PC cultures. Glutamate-induced cell death of 58Q PC cultures was attenuated by dantrolene, a clinically relevant RyanR inhibitor and Ca(2+) stabilizer. In whole animal experiments, we demonstrated that long-term feeding of SCA1-58Q mice with dantrolene alleviated age-dependent motor deficits (quantified in beam-walk and rotarod assays) and reduced PC loss observed in untreated SCA2-58Q mice by 12 months of age (quantified by stereology). Results of our studies indicate that disturbed neuronal Ca(2+) signaling may play an important role in SCA2 pathology and also suggest that the RyanR constitutes a potential therapeutic target for treatment of SCA2 patients.

Deranged calcium signaling and neurodegeneration in spinocerebellar ataxia type 3.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is an autosomal-dominant neurodegenerative disorder caused by a polyglutamine expansion in ataxin-3 (ATX3; MJD1) protein. In biochemical experiments, we demonstrate that mutant ATX3(exp) specifically associated with the type 1 inositol 1,4,5-trisphosphate receptor (InsP(3)R1), an intracellular calcium (Ca(2+)) release channel. In electrophysiological and Ca(2+) imaging experiments, we show that InsP(3)R1 was sensitized to activation by InsP(3) in the presence of mutant ATX3(exp). We found that feeding SCA3-YAC-84Q transgenic mice with dantrolene, a clinically relevant stabilizer of intracellular Ca(2+) signaling, improved their motor performance and prevented neuronal cell loss in pontine nuclei and substantia nigra regions. Our results indicate that deranged Ca(2+) signaling may play an important role in SCA3 pathology and that Ca(2+) signaling stabilizers such as dantrolene may be considered as potential therapeutic drugs for treatment of SCA3 patients.

Familial Alzheimer disease-linked mutations specifically disrupt Ca2+ leak function of presenilin 1.

Mutations in presenilins are responsible for approximately 40% of all early-onset familial Alzheimer disease (FAD) cases in which a genetic cause has been identified. In addition, a number of mutations in presenilin-1 (PS1) have been suggested to be associated with the occurrence of frontal temporal dementia (FTD). Presenilins are highly conserved transmembrane proteins that support cleavage of the amyloid precursor protein by gamma-secretase. Recently, we discovered that presenilins also function as passive ER Ca(2+) leak channels. Here we used planar lipid bilayer reconstitution assays and Ca(2+) imaging experiments with presenilin-null mouse embryonic fibroblasts to analyze ER Ca(2+) leak function of 6 FAD-linked PS1 mutants and 3 known FTD-associated PS1 mutants. We discovered that L166P, A246E, E273A, G384A, and P436Q FAD mutations in PS1 abolished ER Ca(2+) leak function of PS1. In contrast, A79V FAD mutation or FTD-associated mutations (L113P, G183V, and Rins352) did not appear to affect ER Ca(2+) leak function of PS1 in our experiments. We validated our findings in Ca(2+) imaging experiments with primary fibroblasts obtained from an FAD patient possessing mutant PS1-A246E. Our results indicate that many FAD mutations in presenilins are loss-of-function mutations affecting ER Ca(2+) leak activity. In contrast, none of the FTD-associated mutations affected ER Ca(2+) leak function of PS1, indicating that the observed effects are disease specific. Our observations are consistent with the potential role of disturbed Ca(2+) homeostasis in Alzheimer disease pathogenesis.

Presenilins form ER Ca2+ leak channels, a function disrupted by familial Alzheimer's disease-linked mutations.

Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder. Mutations in presenilins 1 and 2 (PS1 and PS2) account for approximately 40% of familial AD (FAD) cases. FAD mutations and genetic deletions of presenilins have been associated with calcium (Ca(2+)) signaling abnormalities. We demonstrate that wild-type presenilins, but not PS1-M146V and PS2-N141I FAD mutants, can form low-conductance divalent-cation-permeable ion channels in planar lipid bilayers. In experiments with PS1/2 double knockout (DKO) mouse embryonic fibroblasts (MEFs), we find that presenilins account for approximately 80% of passive Ca(2+) leak from the endoplasmic reticulum. Deficient Ca(2+) signaling in DKO MEFs can be rescued by expression of wild-type PS1 or PS2 but not by expression of PS1-M146V or PS2-N141I mutants. The ER Ca(2+) leak function of presenilins is independent of their gamma-secretase activity. Our data suggest a Ca(2+) signaling function for presenilins and provide support for the "Ca(2+) hypothesis of AD."

The PDAPP mouse model of Alzheimer's disease: locus coeruleus neuronal shrinkage.

Alzheimer's disease is characterized by neuronal degeneration in the cerebral cortex and hippocampus and subcortical neuronal degeneration in such nuclei as the locus coeruleus (LC). Transgenic mice overexpressing mutant human amyloid precursor protein V717F, PDAPP mice, develop several Alzheimer's disease-like lesions. The present study sought to determine whether there is also loss of LC noradrenergic neurons or evidence of degenerative changes in these animals. PDAPP hemizygous and wild-type littermate control mice were examined at 23 months of age, at a time when there are numerous amyloid-beta (Abeta) plaques in the neocortex and hippocampus. Tissue sections were stained immunohistochemically with an antibody against tyrosine hydroxylase (TH) to identify LC neurons. Computer imaging procedures were used to count the TH-immunoreactive somata in sections through the rostral-caudal extent of the nucleus. There was no loss of LC neurons in the hemizygous mice. In a second experiment, homozygous PDAPP and wild-type mice were examined, at 2 months and 24 months of age. Again there was no age-related loss of neurons in the homozygous animals. In the portion of the LC where neurons reside that project to the cortex and hippocampus, however, the neurons were decreased in size selectively in the 24-month-old transgenic animals. These data indicate that overt LC cell loss does not occur following abundant overexpression of Abeta peptide. However, the selective size reduction of the LC neuronal population projecting to cortical and hippocampal regions containing Abeta-related neuropathology implies that these cells may be subjected to a retrograde-mediated stress.

Inverse relationship between the contents of neuromelanin pigment and the vesicular monoamine transporter-2: human midbrain dopamine neurons.

The dopaminergic neurons in the ventral substantia nigra (SN) are significantly more vulnerable to degeneration in Parkinson's disease (PD) than the dopaminergic neurons in the ventral tegmental area (VTA). The ventral SN neurons also contain significantly more neuromelanin pigment than the dopaminergic neurons in the VTA. In vitro data indicate that neuromelanin pigment is formed from the excess cytosolic catecholamine that is not accumulated into synaptic vesicles by the vesicular monoamine transporter-2 (VMAT2). By using quantitative immunohistochemical methods in human postmortem brain, we sought to examine the relative contents of VMAT2 within neurons that contain different amounts of neuromelanin pigment. The immunostaining intensity (ISI) was measured for VMAT2 and also for the rate-limiting enzyme for the synthesis of dopamine, tyrosine hydroxylase (TH). ISI measures were taken from the ventral SN region where neurons are most vulnerable to degeneration in PD, nigrosome-1 (N1); from the ventral SN region where cells are moderately vulnerable to degeneration in PD, the matrix (M); and from VTA neurons near the exit of the third nerve (subregion III). The data indicate that 1) subregion III neurons have significantly higher levels of VMAT2 ISI compared with N1 neurons (more than twofold) and M neurons (45%); 2) there is an inverse relationship between VMAT2 ISI and neuromelanin pigment in the N1 and III neurons; 3) there is an inverse relationship between VMAT2 ISI and the vulnerability to degeneration in PD in the N1, M, and III subregions; and 4) neurons with high VMAT2 ISI also have high TH ISI. These data support the hypothesis that midbrain dopaminergic neurons that synthesize greater amounts of dopamine have more vesicular storage capacity for action potential-induced release of transmitter and that the ventral SN neurons accumulate the most neuromelanin pigment, in part because they have the least VMAT2 protein.