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Systemic Central Nervous System (CNS)-targeted Delivery of Neuropeptide Y (NPY) Reduces Neurodegeneration and Increases Neural Precursor Cell Proliferation in a Mouse Model of Alzheimer Disease*

Open AccessPublished:November 30, 2015DOI:https://doi.org/10.1074/jbc.M115.678185
      Neuropeptide Y (NPY) is one of the most abundant protein transmitters in the central nervous system with roles in a variety of biological functions including: food intake, cardiovascular regulation, cognition, seizure activity, circadian rhythms, and neurogenesis. Reduced NPY and NPY receptor expression is associated with numerous neurodegenerative disorders including Alzheimer disease (AD). To determine whether replacement of NPY could ameliorate some of the neurodegenerative and behavioral pathology associated with AD, we generated a lentiviral vector expressing NPY fused to a brain transport peptide (apoB) for widespread CNS delivery in an APP-transgenic (tg) mouse model of AD. The recombinant NPY-apoB effectively reversed neurodegenerative pathology and behavioral deficits although it had no effect on accumulation of Aβ. The subgranular zone of the hippocampus showed a significant increase in proliferation of neural precursor cells without further differentiation into neurons. The neuroprotective and neurogenic effects of NPY-apoB appeared to involve signaling via ERK and Akt through the NPY R1 and NPY R2 receptors. Thus, widespread CNS-targeted delivery of NPY appears to be effective at reversing the neuronal and glial pathology associated with Aβ accumulation while also increasing NPC proliferation. Overall, increased delivery of NPY to the CNS for AD might be an effective therapy especially if combined with an anti-Aβ therapeutic.

      Introduction

      Neuropeptide Y (NPY),
      The abbreviations used are: NPY, neuropeptide Y; AD, Alzheimer disease; apoB, apolipoprotein B; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; PCNA, proliferating cell nuclear antigen; DCX, doublecortin; GFAP, glial fibrillary acetic protein; LV, lentiviral vectors; tg, transgenic.
      one of the most abundant neuropeptides in the central and peripheral nervous system is synthesized in neurons and transported to pre- and post-synaptic vesicles where it is secreted (
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      ). NPY is produced as prepro-enzyme that is processed first by removal of the secretory signal in the endoplasmic reticulum to produce the pro-NPY and then by the peptidase convertase to generate NPY(1–39). Further editing by peptidase enzymes generates the mature NPY(1–36) (
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      This mature form binds primarily to three different receptors in the rodent brain, NPY R1, NPY R2, and NPY R5; which are all seven transmembrane receptors (
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      ). Upon binding to the receptor, signaling pathways involve the activation of the Gi/Go receptors and inhibition of cAMP synthesis followed by signaling through protein kinase C, mitogen-activated protein kinase (MAPK), inositol trisphosphate, and extracellular signal related kinase (ERK) (
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      NPY is associated with a variety of biologic functions including: food intake, cardiovascular regulation, cognition, seizure activity, circadian rhythms, and neurogenesis (
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      ). Alterations in NPY have been associated with many neurodegenerative disorders including Downs syndrome (
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      ) immunoreactivity in the hippocampus of AD patients and NPY accumulation in dystrophic neurites around the amyloid plaques (
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      ). In addition, reduced levels of NPY in the plasma (
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      • Kai T.
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      ) and cerebrospinal fluid (
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      • Edvinsson L.
      • Minthon L.
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      • Gustafson L.
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      ) have been found correlating with the onset of AD. Thus reduction in NPY may play a role in pathology of AD or may be a secondary event associated with AD. In APP-tg models of AD the neuronal network dysfunction is associated with NPY alterations in the limbic system (
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      ) and intracerebral infusion of C terminus NPY fragments has been shown to be neuroprotective (
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      The pathogenesis of AD is associated with progressive accumulation of Aβ oligomers leading to synaptic loss, neuronal dysfunction, and death (
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      ). In addition, increased pro-inflammatory cytokine release that contributes to neuronal cell loss (
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      ). The mThy1-APP-tg mouse model (line 41) that our group developed (
      • Rockenstein E.
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      • Masliaha E.
      Early formation of mature amyloid-β proteins deposits in a mutant APP transgenic model depends on levels of Aβ1–42.
      ) recapitulates some of the aspects of this disease and has been useful for study of the pathogenesis of synaptic damage (
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      ), and behavioral deficits (
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      Thy1-hAPP(Lond/Swe+) mouse model of Alzheimer's disease displays broad behavioral deficits in sensorimotor, cognitive and social function.
      ). For example, we have previously shown that intracerebral infusion of the NPY C terminus fragments into the APP-tg mouse model of AD ameliorates the behavioral and neurodegenerative pathology in this model (
      • Rose J.B.
      • Crews L.
      • Rockenstein E.
      • Adame A.
      • Mante M.
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      Neuropeptide Y fragments derived from neprilysin processing are neuroprotective in a transgenic model of Alzheimer's disease.
      ).
      In this study we wanted to develop a more long lasting, systemic delivery of NPY targeted to the brain to determine whether this could ameliorate the neurodegenerative pathology and deficits triggered by Aβ accumulation. For this reason, we engineered a novel recombinant NPY with a blood-brain barrier transport tag. We have previously shown that fusion proteins containing a 38-aa peptide derived from apolipoprotein B (apoB) that recognizes the LDL-R in endothelial cells is capable of shuttling proteins into the CNS (
      • Spencer B.
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      • Spencer B.
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      ,
      • Spencer B.
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      • Rockenstein E.
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      A neuroprotective brain-penetrating endopeptidase fusion protein ameliorates Alzheimer disease pathology and restores neurogenesis.
      • Spencer B.J.
      • Verma I.M.
      Targeted delivery of proteins across the blood-brain barrier.
      ). This recombinant NPY was delivered by lentiviral vector via intraperitoneal injection to the liver for continuous expression and distribution in the blood. Using this method, we were able to show widespread distribution of NPY across the brain, reversal of neurodegeneration, and improved memory in the APP-tg mice. Furthermore, we showed increased proliferation of neural precursor cells in the subgranular zone of the hippocampus. NPY-apoB signaled via ERK and Akt through the NPY R1 and R2, respectively. Thus, widespread delivery of the brain-targeted recombinant NPY might represent a novel therapeutic for AD and other neurodegenerative diseases that show reduction in NPY.

      Discussion

      We report here the construction and characterization of a lentivirus expressing a novel brain targeting and penetrating the NPY protein for widespread delivery to the CNS following systemic administration. NPY-apoB was taken up by neurons across the hippocampus ameliorating neurodegeneration and increasing proliferation of neural precursor cells in the subgranular zone of the dentate gyrus. NPY-apoB signaling occurred through both NPY R1 and NPY R2 receptors. Although increased NPC proliferation was observed, there was little to no increase in basal levels of neuronal differentiation in the hippocampus. Although increased NPY-apoB trafficking to the CNS did not reduce Aβ accumulation, there was an increase in neurogenesis and synaptic density that was accompanied by behavioral improvements. Systemic delivery of the fusion NPY-apoB to the CNS may prove to be a practical approach for replacing the loss of this neuropeptide often observed in AD and other neurodegenerative diseases.
      We observed increased proliferation of NPCs in the subgranular zone but failed to observe an increase in differentiation of these new cells into the hippocampus. It has been previously reported that NPY promotes self-renewal of neural progenitor cells in the subgranular zone of the dentate gyrus (
      • Howell O.W.
      • Silva S.
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      • Zaben M.
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      • McKhann 2nd, G.
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      • Laskowski A.
      • Gray W.P.
      Neuropeptide Y is important for basal and seizure-induced precursor cell proliferation in the hippocampus.
      ); however, it only induces neuronal differentiation in the subventricular zone where it fails to promote self-renewal (
      • Agasse F.
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      • Kristiansen H.
      • Christiansen S.H.
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      • Silva B.
      • Grade S.
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      Neuropeptide Y promotes neurogenesis in murine subventricular zone.
      ,
      • Thiriet N.
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      • Guégan C.
      • Vallette F.
      • Cadet J.L.
      • Jaber M.
      • Malva J.O.
      • Coronas V.
      NPY promotes chemokinesis and neurogenesis in the rat subventricular zone.
      ). In fact, previous reports have shown that the increase in proliferation in the subgranular zone is through the NPY R1 receptor via the ERK pathway (
      • Howell O.W.
      • Doyle K.
      • Goodman J.H.
      • Scharfman H.E.
      • Herzog H.
      • Pringle A.
      • Beck-Sickinger A.G.
      • Gray W.P.
      Neuropeptide Y stimulates neuronal precursor proliferation in the post-natal and adult dentate gyrus.
      ,
      • Howell O.W.
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      • Herzog H.
      • Sundstrom L.E.
      • Beck-Sickinger A.
      • Gray W.P.
      Neuropeptide Y is neuroproliferative for post-natal hippocampal precursor cells.
      ,
      • Baptista S.
      • Bento A.R.
      • Gonçalves J.
      • Bernardino L.
      • Summavielle T.
      • Lobo A.
      • Fontes-Ribeiro C.
      • Malva J.O.
      • Agasse F.
      • Silva A.P.
      Neuropeptide Y promotes neurogenesis and protection against methamphetamine-induced toxicity in mouse dentate gyrus-derived neurosphere cultures.
      ); a receptor that we show in this report is activated by our recombinant NPY-apoB consistent with these previous findings. Although we did not investigate the subventricular zone in this study due its relevance to AD, it would be interesting to examine it in the future.
      NPY R1 is expressed on astrocytes in the hippocampus (
      • St-Pierre J.A.
      • Nouel D.
      • Dumont Y.
      • Beaudet A.
      • Quirion R.
      Sub-population of cultured hippocampal astrocytes expresses neuropeptide Y Y(1) receptors.
      ) and NPY R1 and NPY R2 are both expressed on microglia in the hippocampus. Interestingly, although NPY R2 activation has been shown to play a role in microglia survival in methamphetamine-induced microglia cell death (
      • Gonçalves J.
      • Ribeiro C.F.
      • Malva J.O.
      • Silva A.P.
      Protective role of neuropeptide Y Y(2) receptors in cell death and microglial response following methamphetamine injury.
      ), NPY R1 receptor activation has been shown to inhibit microglia cell activation (
      • Ferreira R.
      • Xapelli S.
      • Santos T.
      • Silva A.P.
      • Cristóvão A.
      • Cortes L.
      • Malva J.O.
      Neuropeptide Y modulation of interleukin-1β (IL-1β)-induced nitric oxide production in microglia.
      ) and the production of the pro-inflammatory cytokine IL-1β as well as downstream components such as iNOS (
      • Ferreira R.
      • Xapelli S.
      • Santos T.
      • Silva A.P.
      • Cristóvão A.
      • Cortes L.
      • Malva J.O.
      Neuropeptide Y modulation of interleukin-1β (IL-1β)-induced nitric oxide production in microglia.
      ). Inflammation and increased gliosis is a hallmark of AD and is observed in the APP-tg model used in this study (
      • Spencer B.
      • Verma I.
      • Desplats P.
      • Morvinski D.
      • Rockenstein E.
      • Adame A.
      • Masliah E.
      A neuroprotective brain-penetrating endopeptidase fusion protein ameliorates Alzheimer disease pathology and restores neurogenesis.
      ). Delivery of the NPY-apoB to the brain resulted in widespread reduction in astrogliosis consistent with the finding that NPY is able to repress neuroinflammatory responses and neurodegeneration, thus down-regulating a key component of the pathology associated with AD. Interestingly, we did not see a change in the numbers of microglial cells in APP-tg mice treated with NPY-apoB. Thus, activation of both NPY R1 and NPY R2 signaling pathways in microglia cells in the hippocampus could reduce inflammation and also does not affect microglia proliferation.
      In addition to regulating neurogenesis and astrogliosis in the CNS, NPY regulates circadian rhythms and food intake. Many neurodegenerative diseases, including AD, manifest with alterations in circadian rhythms (
      • Angelucci F.
      • Gelfo F.
      • Fiore M.
      • Croce N.
      • Mathé A.A.
      • Bernardini S.
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      The effect of neuropeptide Y on cell survival and neurotrophin expression in in-vitro models of Alzheimer's disease.
      ,
      • Corvino V.
      • Marchese E.
      • Podda M.V.
      • Lattanzi W.
      • Giannetti S.
      • Di Maria V.
      • Cocco S.
      • Grassi C.
      • Michetti F.
      • Geloso M.C.
      The neurogenic effects of exogenous neuropeptide Y: early molecular events and long-lasting effects in the hippocampus of trimethyltin-treated rats.
      ,
      • Musiek E.S.
      Circadian clock disruption in neurodegenerative diseases: cause and effect?.
      ) and one prominent clinical feature of AD is weight loss correlating with disease severity (
      • White H.
      • Pieper C.
      • Schmader K.
      The association of weight change in Alzheimer's disease with severity of disease and mortality: a longitudinal analysis.
      ). In fact, several studies have reported that one of the earliest symptoms of AD, manifesting in pre-clinical mild cognitive impairment, may be early weight loss (
      • Barrett-Connor E.
      • Edelstein S.L.
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      • Wiederholt W.C.
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      • Buchman A.S.
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      • Evans D.A.
      • Bennett D.A.
      Change in body mass index and risk of incident Alzheimer disease.
      ,
      • Stewart R.
      • Masaki K.
      • Xue Q.L.
      • Peila R.
      • Petrovitch H.
      • White L.R.
      • Launer L.J.
      A 32-year prospective study of change in body weight and incident dementia: the Honolulu-Asia Aging Study.
      • Johnson D.K.
      • Wilkins C.H.
      • Morris J.C.
      Accelerated weight loss may precede diagnosis in Alzheimer disease.
      ). Although neither weight nor circadian rhythms were monitored in this short-term study, the delivery of systemic NPY-apoB may be beneficial to these other symptoms of neurodegenerative diseases.
      We previously reported that the extracellular endopeptidase neprilysin cleaves the full-length NPY generating a prominent C-terminal fragment composing amino acids 21–36 and 31–36 (
      • Rose J.B.
      • Crews L.
      • Rockenstein E.
      • Adame A.
      • Mante M.
      • Hersh L.B.
      • Gage F.H.
      • Spencer B.
      • Potkar R.
      • Marr R.A.
      • Masliah E.
      Neuropeptide Y fragments derived from neprilysin processing are neuroprotective in a transgenic model of Alzheimer's disease.
      ,
      • Spencer B.
      • Verma I.
      • Desplats P.
      • Morvinski D.
      • Rockenstein E.
      • Adame A.
      • Masliah E.
      A neuroprotective brain-penetrating endopeptidase fusion protein ameliorates Alzheimer disease pathology and restores neurogenesis.
      ). This C-terminal fragment of NPY increases neuroviability and neuroproliferation in the APP-tg mice (
      • Rose J.B.
      • Crews L.
      • Rockenstein E.
      • Adame A.
      • Mante M.
      • Hersh L.B.
      • Gage F.H.
      • Spencer B.
      • Potkar R.
      • Marr R.A.
      • Masliah E.
      Neuropeptide Y fragments derived from neprilysin processing are neuroprotective in a transgenic model of Alzheimer's disease.
      ,
      • Spencer B.
      • Verma I.
      • Desplats P.
      • Morvinski D.
      • Rockenstein E.
      • Adame A.
      • Masliah E.
      A neuroprotective brain-penetrating endopeptidase fusion protein ameliorates Alzheimer disease pathology and restores neurogenesis.
      ). In this report, we showed that systemic delivery of the NPY-apoB increased CNS concentrations of this C-terminal NPY fragment.
      The recombinant NPY-apoB protein produced from the lentiviral platform contains a V5 epitope tag and the 38-amino acid ApoB LDL-receptor binding domain slightly increasing the size of the NPY protein over the size of the recombinant vector-produced NPY-V5 protein (Fig. 1B). Because this size difference was so small, this was observed only on gels where sufficient protein separation was allowed to occur. The in vivo immunoblot analysis of recombinant NPY-V5 and NPY-V5-apoB shows the size separation on the blot probed with the anti-V5 antibody; however, on a separately run gel that was probed for full-length NPY and the CT-NPY, insufficient size separation generates the appearance of similar protein size (Fig. 3D).
      Although NPY-apoB was effective at reversing the neuronal pathology and behavioral deficits as well as increasing proliferation of neural precursor cells, there was no effect on the accumulation of Aβ in the CNS of APP-tg mice. Thus, NPY-apoB might be an effective therapeutic; however, one that would be most effective with an anti-Aβ therapy in combination. Passive immunization (
      • Spencer B.
      • Masliah E.
      Immunotherapy for Alzheimer's disease: past, present and future.
      ) and CNS delivery of Aβ proteases (
      • Spencer B.
      • Marr R.A.
      • Gindi R.
      • Potkar R.
      • Michael S.
      • Adame A.
      • Rockenstein E.
      • Verma I.M.
      • Masliah E.
      Peripheral delivery of a CNS targeted, metallo-protease reduces Aβ toxicity in a mouse model of Alzheimer's disease.
      ,
      • Spencer B.
      • Verma I.
      • Desplats P.
      • Morvinski D.
      • Rockenstein E.
      • Adame A.
      • Masliah E.
      A neuroprotective brain-penetrating endopeptidase fusion protein ameliorates Alzheimer disease pathology and restores neurogenesis.
      ) have been effective at reducing the accumulation of Aβ in the brain and may be effective in combination with CNS-targeted NPY-apoB at treating the many symptoms associated with AD.

      Author Contributions

      B. S. and E. M. conceived of the study and designed experiments. B. S., E. M., and R. P. drafted the manuscript. B. S., R. P., J. M., I. T., A. A., and E. R. performed experiment and analyzed data. E. M. completed statistical analysis of data. All authors read and approved the final manuscript.

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