Differential localization of Rho GTPases in live cells: regulation by hypervariable regions and RhoGDI binding.

Determinants of membrane targeting of Rho proteins were investigated in live cells with green fluorescent fusion proteins expressed with or without Rho-guanine nucleotide dissociation inhibitor (GDI)alpha. The hypervariable region determined to which membrane compartment each protein was targeted. Targeting was regulated by binding to RhoGDI alpha in the case of RhoA, Rac1, Rac2, and Cdc42hs but not RhoB or TC10. Although RhoB localized to the plasma membrane (PM), Golgi, and motile peri-Golgi vesicles, TC10 localized to PMs and endosomes. Inhibition of palmitoylation mislocalized H-Ras, RhoB, and TC10 to the endoplasmic reticulum. Although overexpressed Cdc42hs and Rac2 were observed predominantly on endomembrane, Rac1 was predominantly at the PM. RhoA was cytosolic even when expressed at levels in vast excess of RhoGDI alpha. Oncogenic Dbl stimulated translocation of green fluorescent protein (GFP)-Rac1, GFP-Cdc42hs, and GFP-RhoA to lamellipodia. RhoGDI binding to GFP-Cdc42hs was not affected by substituting farnesylation for geranylgeranylation. A palmitoylation site inserted into RhoA blocked RhoGDI alpha binding. Mutations that render RhoA, Cdc42hs, or Rac1, either constitutively active or dominant negative abrogated binding to RhoGDI alpha and redirected expression to both PMs and internal membranes. Thus, despite the common essential feature of the CAAX (prenylation, AAX tripeptide proteolysis, and carboxyl methylation) motif, the subcellular localizations of Rho GTPases, like their functions, are diverse and dynamic.

The Interplay Between Apolipoprotein E4 and the Autophagic-Endocytic-Lysosomal Axis.

Since its discovery as a genetic risk factor for Alzheimer's disease, the APOE4 allele has been linked to the majority of the pathological findings associated with the disease progression. These include abnormalities of the endocytic, autophagic, and lysosomal machineries, which begin at the most early stages of Alzheimer's disease development. Considering that these three vesicular systems share common features and, in fact, comprise an interconnected cargo-trafficking and degradation network, some of the effects of APOE4 are interrelated, while others are system-specific. In turn, APOE4-driven impairments of endocytosis, autophagy, and lysosomal activity influence various aspects of Alzheimer's disease pathology, ranging from Aß generation and clearance to neuronal loss and cognitive deficits. This review discusses the detrimental effects of APOE4 on the endocytic-autophagic-lysosomal axis in the context of Alzheimer's disease, as well as the various mechanisms underlying them.

Transforming the Perioperative Treatment Paradigm in Non-Metastatic RCC-A Possible Path Forward.

In 2017, there is no adjuvant systemic therapy proven to increase overall survival in non-metastatic renal cell carcinoma (RCC). The anti-PD-1 antibody nivolumab improves overall survival in metastatic treatment refractory RCC and is generally tolerable. Mouse solid tumor models have revealed a benefit with a short course of neoadjuvant PD-1 blockade compared to adjuvant therapy. Two ongoing phase 2 studies of perioperative nivolumab in RCC patients have shown preliminary feasibility and safety with no surgical delays or complications. The recently opened PROSPER RCC trial (A Phase 3 RandOmized Study Comparing PERioperative Nivolumab vs. Observation in Patients with Localized Renal Cell Carcinoma Undergoing Nephrectomy; EA8143) will examine if the addition of perioperative nivolumab to radical or partial nephrectomy can improve clinical outcomes in patients with high risk localized and locally advanced RCC. With the goal of increasing cure and recurrence-free survival (RFS) rates in non-metastatic RCC, we are executing a three-pronged, multidisciplinary approach of presurgical priming with nivolumab followed by resection and adjuvant PD-1 blockade. We plan to enroll 766 patients with clinical stage ≥T2 or node positive M0 RCC of any histology in this global, randomized, unblinded, phase 3 National Clinical Trials Network study. The investigational arm will receive two doses of nivolumab 240 mg IV prior to surgery followed by adjuvant nivolumab for 9 months. The control arm will undergo the current standard of care: surgical resection followed by observation. Patients are stratified by clinical T stage, node positivity, and histology. The trial is powered to detect a 14.4% absolute benefit in the primary endpoint of RFS from the ASSURE historical control of 55.8% to 70.2% at 5 years (HR = 0.70). The study is also powered to detect a significant overall survival benefit (HR 0.67). Key safety, feasibility, and quality of life endpoints are incorporated. PROSPER RCC exemplifies team science with a host of planned correlative work to investigate the impact of the baseline immune milieu and changes after neoadjuvant priming on clinical outcomes.

Antioxidant mechanisms in apolipoprotein E deficient mice prior to and following closed head injury.

Apolipoprotein E deficient mice have distinct memory deficits and neurochemical derangements and their recovery from closed head injury is impaired. In the present study, we examined the possibility that the neuronal derangements of apolipoprotein E deficient mice are associated with oxidative stress, which in turn affects their ability to recover from close head injury. It was found that brain phospholipid levels in apolipoprotein E deficient mice are lower than those of the controls (55+/-15% of control, P<0. 01), that the cholesterol levels of the two mice groups are similar and that the levels of conjugated dienes of the apolipoprotein E deficient mice are higher than those of control mice (132+/-15% of P<0.01). Brains of apolipoprotein E deficient mice had higher Mn-superoxide dismutase (134+/-7%), catalase (122+/-8%) and glutathione reductase (167+/-7%) activities than control (P<0.01), whereas glutathione peroxidase activity and the levels of reduced glutathione and ascorbic acid were similar in the two mouse groups. Closed head injury increased catalase and glutathione peroxidase activities in both mouse groups, whereas glutathione reductase increased only in control mice. The superoxide dismutase activity was unaffected in both groups. These findings suggest that the antioxidative metabolism of apolipoprotein E deficient mice is altered both prior to and following head injury and that antioxidative mechanisms may play a role in mediating the neuronal maintenance and repair derangements of the apolipoprotein E deficient mice.

M1 muscarinic receptors block caspase activation by phosphoinositide 3-kinase- and MAPK/ERK-independent pathways.

When PC12 cells are deprived of trophic support they undergo apoptosis. We have previously shown that survival of trophic factor-deprived PC12M1 cells can be promoted by activation of the G protein-coupled muscarinic receptors. The mechanism whereby muscarinic receptors inhibit apoptosis is poorly understood. In the present study we investigated this mechanism by examining the effect of muscarinic receptor activation on the serum deprivation-induced activity of key players in apoptosis, the caspases, in PC12M1 cells. The results showed that m1 muscarinic activation inhibits caspase activity induced by serum deprivation. This effect appeared to be caused by the prevention of activation of caspases such as caspase-2 and caspase-3, and not by the inhibition of existing activity. Muscarinic receptor activation also stimulated the mitogen-activated protein kinase/extracellular signaling-regulated kinase (MAPK/ERK) and phosphoinositide (PI) 3-kinase signaling pathways. The PI 3-kinase pathway inhibitors wortmannin and LY294002, as well as the MAPK/ERK pathway PD98059 inhibitor, did not however suppress the inhibitory effect of the muscarinic receptors on caspase activity. The results therefore suggested that the muscarinic survival effect is mediated by a pathway that leads to caspase inhibition by MAPK/ERK- and PI 3-kinase-independent signaling cascades.

Cationic defensins arise from charge-neutralized propeptides: a mechanism for avoiding leukocyte autocytotoxicity?

Defensins, small cationic polypeptides with antimicrobial and cytotoxic properties, are among the principal constituents of cytoplasmic granules of mammalian neutrophils and certain macrophages. To identify conserved structural features of defensin precursors that may be important for their targeting to cytoplasmic granules or for prevention of autocytotoxicity, we isolated and sequenced three neutrophil-specific rabbit defensin cDNAs that code for preproprotein precursors to the mature defensins NP-3a, NP-4, and NP-5. The preprodefensins NP-3a, NP-4, and NP-5, like the previously characterized preprodefensins, lack consensus sequences for N-linked glycosylation, suggesting that defensins are targeted to lysosome-like granules by a mechanism not dependent on the mannose-6-phosphate receptor. Analysis of all seven known myeloid prodefensins revealed a structure wherein an anionic propiece neutralizes the cationicity of the mature peptide. Because defensins apparently require cationic epitopes for cell membrane permeabilization and cytotoxicity, charge neutralization of mature peptides by their anionic propieces may prevent autocytotoxicity during defensin synthesis and processing.

Antibodies to different isoforms of the heavy neurofilament protein (NF-H) in normal aging and Alzheimer's disease.

Sera of normal controls and of patients with neurological diseases contain antineurofilament antibodies. Recent studies suggest that biochemically and immunologically distinct subclasses of neurofilaments occur in different types of neurons. Alzheimer's disease (AD), the major cause of dementia, is associated with a marked degeneration of brain cholinergic neurons. In the present work we characterized the repertoire and age dependence of antineurofilament antibodies in normal sera and examined whether the degeneration of cholinergic neurons in AD is associated with serum antibodies directed specifically against the neurofilaments of mammalian cholinergic neurons. This was performed by immunoblot assays utilizing neurofilaments from the purely cholinergic bovine ventral root neurons and from the chemically heterogeneous bovine dorsal root neurons. Antibodies to the heavy neurofilament protein NF-H were detected in normal control sera. Their levels were significantly higher in older (aged 70-79) than in younger (aged 40-59) subjects. These antibodies bound similarly to bovine ventral root and dorsal root NF-H and their NF-H specificity was unchanged during aging. In contrast, the levels of IgG in AD sera that are directed against ventral root cholinergic NF-H were higher than those directed against the chemically heterogeneous dorsal root NF-H. Immunoblot experiments utilizing dephosphorylated ventral root and dorsal root NF-H and chymotryptic fragments of these molecules revealed that AD sera contain a repertoire of antimamalian NF-H IgG. A subpopulation of these antibodies binds to phosphorylated epitopes that are specifically enriched in ventral root cholinergic NF-H and that are located on the carboxy terminal domain of this molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical evidence that acetylcholine release from cholinergic nerve terminals is mostly vesicular.

The nature of the intraterminal compartments from which acetylcholine (ACh) is released following presynaptic stimulation was investigated. This was pursued by examining the effects of the anticholinergic drug 2-(4-phenylpiperidino)cyclohexanol (AH5183) on the release of newly synthesized [3H]ACh and of endogenous ACh from purified cholinergic nerve terminals (synaptosomes) which were isolated from the electric organs of Torpedo. Preincubation of the synaptosomes, with AH5183 (1-10 microM), does not affect either the intraterminal synthesis of [3H]ACh or the uptake of its precursors, but results in a marked inhibition (85%) of the release of the newly synthesized [3H]ACh. However, when AH5183 is added following the accumulation of [3H]ACh in the nerve terminals, it does not affect [3H]ACh release. AH5183 also has no effect on the release of preformed endogenous ACh. These findings, together with the previous in vitro demonstrations that AH5183 is a potent inhibitor of ACh uptake into isolated cholinergic vesicles, suggest that most of the synaptosomal ACh is secreted by a vesicular mechanism.

The structure of neutrophil defensin genes.

Defensins are a family of microbicidal peptides abundant in the granules of mammalian neutrophils, in rabbit alveolar macrophages, and in human and murine intestinal Paneth cells. We cloned and sequenced the genes of three neutrophil-specific defensins. Human HNP-1 and HNP-3 are nearly identical and rabbit NP-3a is closely related. The four known neutrophil-specific defensin genes are strikingly similar in the structure and organization of their three exons and two introns, but the three defensin genes expressed in macrophages (MCP-1 and -2) or Paneth cells (HD-5) are organized differently: HD-5 had only two exons, and MCP-1 and -2 have a comparatively short first intron. The diverse genomic organization of defensin genes may contribute to their cell-specific expression.

Differential inhibition of the release of endogenous and newly synthesized acetylcholine from Torpedo synaptosomes by presynaptic muscarinic receptors.

Activation of Torpedo presynaptic muscarinic acetylcholine (ACh) receptors with the agonist oxotremorine (20 microM) results in the inhibition of Ca2+-dependent release of endogenous ACh from Torpedo synaptosomes. This effect is reversed by the muscarinic antagonist atropine (1 microM) which, by itself, has no effect. In contrast, under the same conditions the amount of newly synthesized radiolabeled [3H]ACh released is not affected by muscarinic ligands. These findings suggest that presynaptic muscarinic inhibition in the Torpedo is due to interference with the mobilization of ACh from a storage pool.

Saturable [D-Ala2, D-Leu5]-enkephalin transport into cholinergic synaptic vesicles.

Incubation of cholinergic synaptic vesicles purified from the electric organ of Torpedo ocellata with radiolabeled [D-Ala2, D-Leu5]-enkephalin results in its accumulation in the vesicles. Kinetic analysis of the initial rates of uptake reveals temperature-dependent saturation kinetics which are best fitted by a single transport system (KT = 12 +/- 2 microM and Vmax = 0.85 +/- 0.24 pmol/mg protein per min). The specific rates and extents of [3H]-[D-Ala2, D-Leu5]-enkephalin uptake, like those of [3H]acetylcholine uptake, are highest in the purified synaptic vesicles fraction. These findings suggest that Torpedo cholinergic synaptic vesicles contain an opioid peptide transporter. The physiological significance of this transporter is discussed in view of the recent observation that Torpedo nerve terminals contain an endogenous enkephalin-like peptide and presynaptic opiate receptors.

The effects of APOE genotype on age at onset and progression of neurodegenerative diseases.

APOE genotype influences the age at onset of some neurodegenerative diseases such as AD and the rate of progression in others such as MS. The authors hypothesize that APOE genotype ubiquitously determines the efficacy of neuronal maintenance and repair in these diseases and that the seemingly divergent clinical effects are due to the stage of disease at which the diagnosis is made. Early diagnosis facilitates the measurement of effects on disease progression rate, whereas late diagnosis results in a marked effect of APOE genotype on disease onset.

Antibodies from Down's syndrome patients bind to the same cholinergic neurofilament protein recognized by Alzheimer's disease antibodies.

Down's syndrome (DS) patients who survive beyond the third decade develop brain lesions characteristic of Alzheimer's disease (AD). Sera of AD patients contain antibodies that bind specifically to the heavy neurofilament protein (NF-H) of Torpedo cholinergic neurons. In the present report, we examined whether the AD-like pathologic changes in DS are associated with the existence of such antibodies. Our findings show that IgG of older DS patients (greater than 30 years) binds to Torpedo cholinergic NF-H more readily than does that of young DS patients (less than 30 years) and age-matched normal controls. In contrast, the extent of binding of IgG from the young and older DS groups to Torpedo and bovine spinal cords NF-H is similar and equal to that of normal controls. These findings suggest that older DS patients, like AD patients, contain anti-NF-H IgG that binds specifically to epitopes highly enriched in Torpedo cholinergic NF-H.

Antibodies to brain antigens following stroke.

Cerebral necrosis following stroke exposes brain antigens to the immune system, potentially initiating an antibody response. The authors measured levels of antibodies to specific neuronal antigens, neurofilaments (NF), and a ubiquitous antigen, cardiolipin (CL), in 45 patients following an acute first-ever stroke, within 48 hours, and 1, 3, and 6 months later. The mean levels of anti-NF antibodies were elevated compared with baseline at 1, 3, and 6, months (p = 0.012, 0.002, and 0.003 by paired t-test). Anti-CL levels did not change significantly.

Antineurofilament antibodies in postpolio syndrome.

We determined the levels of antineurofilament antibodies in 29 patients with postpolio syndrome (PPS), 26 stable postpolio (PP) patients, 22 patients with ALS, and 20 normal controls (NCs). Patients with PPS had higher antibody levels to cholinergic neurofilaments than did all other groups. PP patients and those with ALS had antibody levels similar to those of NCs. The antibody binding level showed no relation to the age of the patients, duration of disease, or motor score.

APOE genotype is a major predictor of long-term progression of disability in MS.

BACKGROUND AND OBJECTIVE: The authors recently reported that the APOE epsilon4 allele is associated with significantly greater progression of disability in a 2-year follow-up of patients with MS. In this study, these findings are substantiated and extended in a much larger group of patients followed for up to 40 years. METHODS: Two hundred five patients with clinically definite MS who were genotyped for the APOE epsilon4 carrier state were included. Groups of patients with (n = 41) and without (n = 164) APOE epsilon4 alleles were compared for latency to expanded disability status scale (EDSS) scores of 4.0 and 6.0 by Kaplan-Meier analysis with the log rank test. The results were adjusted for age at onset and sex by Cox regression analysis. RESULTS: The APOE epsilon4 allele frequency in patients with MS (0.10) was similar to that in the general Israeli population. There was a significant effect of APOE genotype on the latency to reach EDSS 4.0 and 6.0 (p = 0.0002 and p = 0.0006 by two-tailed log rank test). Median latencies were shorter by 12 and 11 years in the APOE epsilon4 group for these outcomes. These results were significant after adjustment for age at onset and sex. CONCLUSIONS: The APOE epsilon4 allele is associated with significantly faster progression of disability in MS. This is the first genetic factor to be identified with a major impact on the progression of disability in this disease.

Metabolism of arachidonic acid and prostaglandins in the Torpedo electric organ: modulation by the presynaptic muscarinic acetylcholine receptor.

We have found that Torpedo electric organ readily synthesizes prostaglandin E2 from both exogenous and endogenous arachidonate and that activation of the presynaptic muscarinic acetylcholine receptor increases the rate of prostaglandin E2 synthesis by inducing the release of tissue arachidonate from its phospholipid pools. The incorporation of radiolabeled arachidonate into tissue phospholipids is slow and Ca2+ independent. However, the electric organ slices readily oxidize the externally added, radiolabeled arachidonate via the cyclo-oxygenase pathway, with prostaglandin E2 being the major product (22 +/- 4% of the initial radioactivity). This process is not affected by either Ca2+ or mepacrine. Torpedo electric organ slices also synthesize prostaglandin E2 from endogenous substrates, and release it into the medium. This process, however, is enhanced by Ca2+ and inhibited by mepacrine. Activation of the Torpedo muscarinic acetylcholine receptor by the agonist oxotremorine results in a dose-dependent atropine-sensitive increase in the synthesis of prostaglandin E2 from endogenous tissue substrates and in the concomitant release of arachidonate into the medium. By contrast, oxotremorine has no effect on either the formation of [14C]prostaglandin E2 from exogenous arachidonate, the incorporation of radiolabeled arachidonate into tissue phospholipids or its liberation from prelabeled slices. These results suggest that activation of the muscarinic acetylcholine receptor induces lipolysis which results in the liberation of endogenous arachidonate and its subsequent conversion to prostaglandin E2.

Decreased density of forebrain cholinergic neurons in experimental autoimmune dementia.

Sera of Alzheimer's disease and Down's syndrome patients contain antibodies which bind specifically to the high molecular weight neurofilament protein of Torpedo cholinergic neurons. We have recently shown that prolonged immunization of rats with this antigen results in the accumulation of IgG in neurons in the septum and hippocampus of the immunized rats and in cognitive impairments. This animal model is termed experimental autoimmune dementia. In the present study we examined whether the anti-cholinergic high molecular weight neurofilament subunit immune response of the experimental autoimmune dementia rats affects forebrain cholinergic neurons. This was performed immunohistochemically utilizing a monoclonal antibody to nerve growth factor receptor, a specific marker of cholinergic neurons in the forebrain. The results obtained revealed significant decreases in the density of cholinergic neurons in the medial septal nucleus and diagonal band of the experimental autoimmune dementia rats. These decreases are specific to the anti-cholinergic high molecular weight neurofilament subunit immune response of the experimental autoimmune dementia rats and are not observed in control rats which were immunized with chemically heterogeneous high molecular weight neurofilament subunit. The decrease in density of forebrain cholinergic neurons in experimental autoimmune dementia rats may mimic pathogenic processes in Alzheimer's disease and supports a role for anti-cholinergic high molecular weight neurofilament subunit antibodies in the degeneration of cholinergic neurons in the disease.

Reversal of presynaptic deficits of apolipoprotein E-deficient mice in human apolipoprotein E transgenic mice.

Apolipoprotein E genotype is an important risk factor of Alzheimer's disease, which is associated with the degeneration of distinct brain neuronal systems. In the present study we employed apolipoprotein E-deficient mice and human apolipoprotein E3 and apolipoprotein E4 transgenic mice on a null mouse apolipoprotein E background, to examine the extent to which distinct brain neuronal systems are affected by apolipoprotein E and the isoform specificity of this effect. This was pursued by histological and autoradiographic measurements utilizing neuron specific presynaptic markers. The results thus obtained revealed significant reductions in the levels of brain cholinergic and noradrenergic nerve terminals in young apolipoprotein E-deficient mice and no changes in brain dopaminergic nerve terminals. These cholinergic and noradrenergic presynaptic derangements were ameliorated similarly in human apolipoprotein E3 and apolipoprotein E4 transgenic mice. In the case of the cholinergic system, this resulted in complete reversal of the presynaptic deficits, whereas in the case of the noradrenergic neurons the amelioration was partial. These findings suggest that brain cholinergic and noradrenergic neurons are markedly more dependent on brain apolipoprotein E than brain dopaminergic neurons and that the isoform specificity of these effects is not apparent at a young age under non-challenged conditions.

Motor and cognitive deficits in apolipoprotein E-deficient mice after closed head injury.

Previous studies suggest that traumatic brain injury is associated with increased risk factor for developing Alzheimer's disease. Furthermore, the extent of the risk seems to be most pronounced in Alzheimer's disease patients who carry the epsilon4 allele of apolipoprotein E, suggesting a connection between susceptibility to head trauma and the apolipoprotein E genotype. Apolipoprotein E-deficient mice provide a useful model for investigating the role of this lipoprotein in neuronal maintenance and repair. In the present study apolipoprotein E-deficient mice and a closed head injury experimental paradigm were used to examine the role of apolipoprotein E in brain susceptibility to head trauma and in neuronal repair. Apolipoprotein E-deficient mice were assessed up to 40 days after closed head injury for neurological and cognitive functions, as well as for histopathological changes in the hippocampus. A neurological severity score used for clinical assessment revealed more severe motor and behavioural deficits in the apolipoprotein E-deficient mice than in the controls, the impairment persisting for at least 40 days after injury. Performance in the Morris water maze, which tests spatial memory, showed a marked learning deficit of the apolipoprotein E-deficient mice when compared with injured controls, which was apparent for at least 40 days. At this time, histopathological examination revealed overt neuronal cell death bilaterally in the hippocampus of the injured apolipoprotein E-deficient mice. The finding that apolipoprotein E-deficient mice exhibit an impaired ability to recover from closed head injury suggests that apolipoprotein E plays an important role in neuronal repair following injury and highlights the applicability of this mouse model to the study of the cellular and molecular mechanisms involved.