Neural Stem Cell Transplant-Induced Effect on Neurogenesis and Cognition in Alzheimer Tg2576 Mice Is Inhibited by Concomitant Treatment with Amyloid-Lowering or Cholinergic α7 Nicotinic Receptor Drugs.

Stimulating regeneration in the brain has the potential to rescue neuronal networks and counteract progressive pathological changes in Alzheimer's disease (AD). This study investigated whether drugs with different mechanisms of action could enhance neurogenesis and improve cognition in mice receiving human neural stem cell (hNSC) transplants. Six- to nine-month-old AD Tg2576 mice were treated for five weeks with the amyloid-modulatory and neurotrophic drug (+)-phenserine or with the partial α7 nicotinic receptor (nAChR) agonist JN403, combined with bilateral intrahippocampal hNSC transplantation. We observed improved spatial memory in hNSC-transplanted non-drug-treated Tg2576 mice but not in those receiving drugs, and this was accompanied by an increased number of Doublecortin- (DCX-) positive cells in the dentate gyrus, a surrogate marker for newly generated neurons. Treatment with (+)-phenserine did however improve graft survival in the hippocampus. An accumulation of α7 nAChR-expressing astrocytes was observed around the injection site, suggesting their involvement in repair and scarring processes. Interestingly, JN403 treatment decreased the number of α7 nAChR-expressing astrocytes, correlating with a reduction in the number of DCX-positive cells in the dentate gyrus. We conclude that transplanting hNSCs enhances endogenous neurogenesis and prevents further cognitive deterioration in Tg2576 mice, while simultaneous treatments with (+)-phenserine or JN403 result in countertherapeutic effects

Neurotrophic and neuroprotective actions of (-)- and (+)-phenserine, candidate drugs for Alzheimer's disease.

Neuronal dysfunction and demise together with a reduction in neurogenesis are cardinal features of Alzheimer's disease (AD) induced by a combination of oxidative stress, toxic amyloid-β peptide (Aβ) and a loss of trophic factor support. Amelioration of these was assessed with the Aβ lowering AD experimental drugs (+)-phenserine and (-)-phenserine in neuronal cultures, and actions in mice were evaluated with (+)-phenserine. Both experimental drugs together with the metabolite N1-norphenserine induced neurotrophic actions in human SH-SY5Y cells that were mediated by the protein kinase C (PKC) and extracellular signal-regulated kinases (ERK) pathways, were evident in cells expressing amyloid precursor protein Swedish mutation (APP(SWE)), and retained in the presence of Aβ and oxidative stress challenge. (+)-Phenserine, together with its (-) enantiomer as well as its N1- and N8-norphenserine and N1,N8-bisnorphenserine metabolites, likewise provided neuroprotective activity against oxidative stress and glutamate toxicity via the PKC and ERK pathways. These neurotrophic and neuroprotective actions were evident in primary cultures of subventricular zone (SVZ) neural progenitor cells, whose neurosphere size and survival were augmented by (+)-phenserine. Translation of these effects in vivo was assessed in wild type and AD APPswe transgenic (Tg2576) mice by doublecortin (DCX) immunohistochemical analysis of neurogenesis in the SVZ, which was significantly elevated by 16 day systemic (+)-phenserine treatment, in the presence of a (+)-phenserine-induced elevation in brain- derived neurotrophic factor (BDNF)

Enhanced doublecortin reactivity in the SVZ of 4–6 months old APPswe mice after (+)-phenserine treatment.

<p>Doublecortin (DCX) immunoreactivity in the subventricular zone (SVZ) in 4–6 months old mice are shown in A and B. Cortical BDNF levels in wt 4–6 months old mice are shown in C. **p<0.01 compared to wt mice treated with saline. Results are expressed as mean ± SEM.</p

Phenserine and primary metabolites exert neuroprotective actions against H₂O₂ and glutamate-induced toxicity in SH-SY5Y cells.

<p>Cells were exposed to 3, 10 or 30 µM (+)-phenserine 24 hr before addition of 30–100 µM H₂O₂ or to 100 mM glutamate in (A), or to (−)-phenserine, (+)-N1-norphenserine, (+)-N8-norphenserine or (+)-N1,N8-bisnorphenserine 24 hr before addition of 100 µM H₂O₂ in (B). In (C), inhibitors of PKC (GF109203X, 2.5 µM), MEK1/2 (U0126, 5 µM) or MEK1 (PD98059, 10 µM) were added to the cells 30 min before adding 30 µM (+)-phenserine. 100 µM H₂O₂ was added after 24 hr and after additional 24 hr incubation, cell proliferation was determined by MTS assay. *P<0.05 **p<0.01, and ***p<0.001 compared to control samples without drug, #p<0.05, ##<i>p</i><0.01 and ###<i>p<</i>0.001 compared to samples with (+)-phenserine only, °°° compared to samples exposed to (+)-phenserine and H₂O₂ (one-way ANOVA followed by Bonferronís multiple comparison post hoc test). Results are expressed as mean ± SEM.</p

Enhanced subventricular progenitor cell survival and increased size of Tg2576 mouse neurospheres after (+)-phenserine exposure.

<p>Representative images show SVZ progenitor cells exposed to vehicle or 0.01 µM (+)-phenserine (A). Effects of the MEK1/2 inhibitor U0126, MEK1 inhibitor PD98059, and PKC inhibitor GF109203X on neurosphere size and neurosphere density in the presence of (+)-phenserine are shown in (B). ***P<0.001 compared to control samples without (+)-phenserine or inhibitor, ##<i>P</i><0.01 and ###<i>P<</i>0.001 compared to samples with (+)-phenserine only (one-way ANOVA followed by Bonferronís multiple comparison post hoc test). The number of neurospheres composed of SVZ progenitor cells, isolated from the lateral (LGE) and medial (MGE) ganglionic eminence at embryonic day 13.5, were measured 7 days after exposure to 0.05, 0.1 and 1 µM (+)-phenserine (C). *p<0.05, **p<0.01, and ***<i>p</i><0.001 compared to samples without (+)-phenserine. Data were analysed with one-way ANOVA followed by Dunnetts post hoc test. Results are expressed as mean ± SEM.</p

Neurotrophic actions retained in APPswe transfected and wt SH-SY5Y cells challenged with Aβ42 and H₂O₂.

<p>Cells were exposed to (+)-phenserine (30 µM) for 24 hr before measuring cell proliferation using MTS assay in (A). 10 or 30 µM (+)-phenserine was added 24 hr before adding 0.1, 1 or 10 µM Aβ42 or 10 µM H₂O₂ before measuring cell proliferation in untransfected SH-SY5Y cells (B). **p<0.01, and ***p<0.001 compared to control samples without drug, ##<i>p</i><0.01 and ###<i>p<</i>0.001 compared to samples with (+)-phenserine only (one-way ANOVA followed by Bonferronís multiple comparison post hoc test). Results are expressed as mean ± SEM.</p

The effects of (+)-phenserine treatment on cell proliferation in the hippocampi of Tg2576 mice.

<p>The number of bromodeoxyuridine-positive (BrdU⁺) cells was increased in hippocampal regions of 4- to 6-month-old Tg2576 mice treated with saline (Sal) or (+)-phenserine (Phe). The number of BrdU⁺ cells was counted in the dentate gyrus (DG) (<b>A</b>), and in the CA1 region (<b>B</b>) in the hippocampi of wild-type (Wt) and Tg2576 mice. Representative images of the DG and CA1 in Tg2576 mice treated with saline (Sal) or (+)-phenserine (Phe) are shown in (<b>C</b>). The number of BrdU⁺ cells in the DG of 15- to 18-month-old Wt and Tg2576 mice treated with saline or (+)-phenserine is shown in Figures (<b>D</b>) and (<b>E</b>). *<i>P</i><0.05 compared to saline-treated Tg2576 mice aged 4–6 or 15–18 months (unpaired t-test). Values are expressed as means ± SEM. Sections were immunolabeled with BrdU (red) and the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, blue) and visualized at 20X magnification.</p

Synaptophysin protein levels in the cerebral cortices of 4- to 6- and 15- to 18-month-old wild-type (Wt, white bars) and Tg2576 mice (black bars) after treatment with saline (Sal) or (+)-phenserine (Phe).

<p>The signals corresponding to the synaptophysin levels were normalized to β-actin in each gel, and a pooled sample (P) was used to control the intergel variability. **<i>P</i><0.01 compared to saline-treated 4- to 6-month-old Tg2576 mice (Mann-Whitney test). Values are expressed as means ± SEM.</p