NGF but not BDNF overexpression protects hippocampal LTP from beta-amyloid-induced impairment

Neuroscience 289 (2015) 114–122

NGF BUT NOT BDNF OVEREXPRESSION PROTECTS HIPPOCAMPAL LTP FROM BETA-AMYLOID-INDUCED IMPAIRMENT A. D. IVANOV, a* G. R. TUKHBATOVA, b S. V. SALOZHIN b AND V. A. MARKEVICH a

NEUROTROPHIC FACTORS AND THEIR FUNCTIONS

a

Laboratory of Neurophysiology of Learning, Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, 5A Butlerova Street, Moscow 117485, Russia

Neurotrophic factors or neurotrophins are essential for neuron development and survival, as well as for synaptic plasticity both in the CNS and the PNS (Thal, 1996; Huang and Reichardt, 2001; Volosin et al., 2006; Mocchetti and Brown, 2008; Conner et al., 2009). Four major neurotrophins were found in the mammalian brain: NGF (nerve growth factor), BDNF (brain-derived neurotrophic factor), NT-3 (neurotrophin-3) and NT-4 (neurotrophin-4). The most widespread of these in the adult brain are BDNF and NGF, whereas NT-3 and NT-4 are much more specialized and less common. In the CNS, the neurotrophins activate two types of receptors: high-affinity Trk receptors (tropomyosine receptor kinase) and low-affinity p75NTR (Counts and Mufson, 2005; Skaper, 2008). Upon binding to their high-affinity receptors, neurotrophins activate three major intracellular signaling cascades essential for synaptic plasticity and cell survival: Ras/ERK, PI3K/Akt and PKC/ PLC cascades (Reichardt, 2006). The p75NTR also activates three cascades but only the NF-kB cascade has a positive effect on neurons, while JNK and RhoA cascades are thought to provoke axon disruption and activate cell death via apoptosis (Huang and Reichardt, 2001; Lu et al., 2005). For many years, neurotrophins were considered as promising candidates for the treatment of various neuropathologies including trauma, lateral sclerosis, Alzheimer’s disease and Parkinson’s disease (Tuszynski et al., 2002). Neuroprotective effects of BDNF were described in various animal models, such as spinal cord injury, focal brain ischemia and excitotoxic neuronal death (Beck et al., 1994; Namiki et al., 2000; Scha¨bitz et al., 2000; Husson et al., 2005; Bemelmans et al., 2006). NGF injection prevents degeneration of cholinergic neurons after fornix leisure or administration of toxins (Williams et al., 1986; Koliatsos et al., 1990; Charles et al., 1996; Cooper et al., 1996; Ruberti et al., 2000; Blesch et al., 2005) and restores the phenotype of cholinergic neurons in a mouse model of Down’s syndrome (Cooper et al., 2001). One of the most extensively studied neuropathologies is Alzheimer’s disease – a devastating disorder associated with synapse loss, memory impairment and degeneration of cholinergic neurons. Sporadic cases of the disease are prevalent although exact nature of their development is still elusive. Beta-amyloid peptide (Ab) is reported to be involved in neuronal degeneration and

b Laboratory of Molecular Neurobiology, Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, 5A Butlerova Street, Moscow 117485, Russia

Abstract—Two major neurotrophic factors, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are involved in a number of physiological processes associated with neuronal growth, survival and plasticity. There are an increasing number of papers demonstrating their ability to serve as neuroprotective molecules under various pathological conditions. At the same time, it remains unclear whether both NGF and BDNF have similar roles under pathological conditions and their effects on the electrophysiological properties of neurons after acute pathogen exposure. In the present paper we investigated the neuroprotective role of these two neurotrophins in a well-characterized model of beta-amyloid peptide (Ab)-dependent impairment of long-term potentiation (LTP). Using lentiviral gene delivery we performed long-term elevation of neurotrophin expression in the dentate gyrus (DG) of rats. One week after virus injection acute brain slices were incubated with beta-amyloid (25–35) for 1 h and afterward in vitro LTP induction was performed in medial perforant path–DG synapses. We demonstrate that chronic elevation of NGF but not BDNF concentration protects LTP induction from betaamyloid action. Further inhibitory analysis suggests that the effect of NGF is mediated by PI3K-signaling cascade. Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Key words: NGF, BDNF, LTP, beta-amyloid, plasticity, neuroprotection.

*Corresponding author. Tel: +7-915-189-57-55. E-mail address: [email protected] (A. D. Ivanov). Abbreviations: Ab, beta-amyloid peptide; ACSF, artificial cerebrospinal fluid; ANOVA, analysis of variance; BDNF, brain-derived neurotrophic factor; CMV, cytomegalovirus; DG, dentate gyrus; DMSO, dimethyl sulfoxide; EGFP, enhansed green fluorescent protein; ELISA, enzyme-linked immunosorbent assay; fEPSPs, field excitatory postsynaptic potentials; hBDNF, human BDNF; HFS, highfrequency stimulation; hNGF, human NGF; HRP, horseradish peroxidase; IRES, internal ribosome entry site; LTP, long-term potentiation; MPP, medial perforant pathway; NGF, nerve growth factor; NT-3, neurotrophin-3; NT-4, neurotrophin-4; PVDF, polyvinylidene difluoride; SDS-PAAG, sodium dodecyl sulfate polyacrylamide gel; TBS-T, Tris-buffered saline-Tween. http://dx.doi.org/10.1016/j.neuroscience.2014.12.063 0306-4522/Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved. 114

A. D. Ivanov et al. / Neuroscience 289 (2015) 114–122

synaptic plasticity disruption during Alzheimer’s disease pathogenesis (Cullen et al., 1997; Chen et al., 2002; Giacchino et al., 2000; Chapman et al., 2001; Freir et al., 2001; Dewachter et al., 2002; Wang et al., 2002; Rowan et al., 2003). Current in vivo and in vitro models of Alzheimer’s disease are based on Ab metabolism corruption or direct Ab application (Larson et al., 1999; Moechars et al., 1999; Chen et al., 2002; Chapman et al., 2001; Dewachter et al., 2002; Wang et al., 2002; Rowan et al., 2003). The possible cross-talk between neurotrophin and amyloid systems in the brain was illustrated in NGF-deprived AD11 transgenic mice. Experimental abrogation of NGF-dependent signaling in AD11 mice leads to accumulation of toxic amyloid fragments (Capsoni et al., 2002) One of the consequences of the elevation of beta-amyloid level is reduction of long-term potentiation (LTP) in medial perforant path (MPP)– dentate gyrus (DG) synapse (Houeland et al., 2010). Interestingly, these abnormalities of LTP induction were region-specific since potentiation at Schaffer collateral– CA1 synapses was unaffected in AD11 mice. This may reflect different cellular vulnerability in pathological conditions since DG granule cells are more sensitive to Ab. To examine whether enhancement of neurotrophindependent signaling may protect DG granule cells’ synaptic plasticity, we used lentiviral-mediated delivery of genes encoding either NGF or BDNF in the DG and then analyzed the amplitude and dynamics of LTP in normal conditions and after Ab impact in vitro. We showed that NGF but not BDNF selectively protect LTP and this action relies on PI3K-dependent signaling.

EXPERIMENTAL PROCEDURES Animals Sixty male P20–P25 Wistar rats (50–70 g) supplied by the ‘‘Stolbovaya’’ Breeding Center (Moscow, Russia) were used in this study. Rats were housed four per cage at an ambient temperature of 22–25 °C and under a day– night cycle 12-h:12-h with free access to food and water. All experiments were performed in accordance with the EC Directive 2010/63/EU on the protection of animals used for scientific purposes and were approved by IHNA Bioethics Commission. All efforts were made to minimize animal suffering and to reduce the number of animals used. Plasmids synthesis and virus preparation Lentiviral vectors encoding human NGF (hNGF) and human BDNF (hBDNF) were used in this study. Clones encoding verified cDNA for hNGF and hBDNF were obtained from OpenBiosystems. NGF and BDNF were amplified with respective pairs of primers containing BamHI sites on the 50 and 30 ends and cloned into pCSC-SP-PW-internal ribosome entry site (IRES)_GFP lentiviral vector kindly provided by Dr. Alan Chen. Successful clones were verified by restriction digestion and sequencing. For virus production we used calcium phosphate transfection of ten 100-mm Petri dishes of

115

HEK293T. The following plasmids were used: 12 lg pLenti, 7.8 lg pMDL, 4.2 lg pVSV-G, and 3 lg pRev. Two supernatants were harvested 24 and 48 h post transfection and concentrated at 50,000g in a CP80WX ultracentrifuge. Pellet was resuspended in OPTI-MEM, aliquoted and stored at 70 °C. Viral titer was checked in serial dilutions on HEK293T cells.

Stereotaxic injections of lentiviral suspension One-month-old rats were randomly divided into 4 groups: naı¨ ve, cytomegalovirus (pCMV)-enhansed green fluorescent protein (EGFP) (cited below as control), pCMV-hNGF-IRES-EGFP (cited below as NGF) and pCMV-BDNF-IRES-EGFP (cited below as BDNF). Rats were anesthetized with chloral hydrate (0.4-g/kg bodyweight) intraperitoneally and fixed in a Kopf stereotaxis. The skalp was cut sagittally and retracted. Injection sites coordinates were measured from the Bregma according to Paxinos and Watson (2005) rat brain atlas. The skull was drilled manually and 2 ll of concentrated titer-matched lentiviral suspension was injected by an automatic nanoinjector into the right DG (2.5 mm AP; 1.5 mm ML; 3.5 mm DV) at a rate of 0.5 ll/min. Animals were allowed to recover 7 days before electrophysiological recordings.

Ab preparation Ab [25–35] and reverse Ab [35–25] (Bachem, Switzerland) were re-suspended in sterile mQ water to obtain a 1 mM solution, aliquoted into microcentrifuge tubes by 10 ll and stored at 20 °C. Before each experiment, a new aliquot of 1 mM Ab was incubated for 12 h at 4 °C. Finally, desired concentrations of Ab were obtained by subsequent dilution of 1 mM Ab in artificial cerebro-spinal fluid (ACSF) up to 20, 50 and 100 nM. Reverse Ab [35–25] was diluted up to 50 nM.

Slice preparation Seven days after viral injections, rats were decapitated. 500-lm transverse brain slices were cut using a Leica VT1200S vibratome in ice-cold cutting solution containing (in mM): 124 NaCl, 3 KCl, 26 NaHCO3, 1.25 NaH2PO4, 2.6 MgSO4, 25 Glucose and 1.3 CaCl2 (saturated with 95% O2 and 5% CO2). Normally, we prepared two slices from one animal brain and then recorded them at the same time, one under ‘‘normal conditions’’ and the other after pre-incubation in Ab. Thus, in each group, every single slice was made from a different animal. Slices were left to recovery for 1 h at room temperature in oxygenated ACSF made of (in mM) 124 NaCl, 3 KCl, 26 NaHCO3, 1.25 NaH2PO4, 1.3 MgSO4, 25 Glucose and 2.5 CaCl2. After recovery slices of the appropriate groups were pre-incubated with Ab, LY294002 or both. For field excitatory postsynaptic potentials (fEPSPs) recording, slices were transferred to recording chambers and continuously perfused at 3–5 ml/min with oxygenated ACSF warmed to 31–32 °C.

116

A. D. Ivanov et al. / Neuroscience 289 (2015) 114–122

Electrophysiology fEPSPs were recorded using standard electrophysiological techniques from the middle part of the granulate layer of the DG with a monopolar glass microelectrode which was filled with ACSF and had average resistance of 3–6 MX. Presynaptic stimulation was applied to the MPP of the DG using a bipolar stimulating electrode made from two isolated nichrome wires (d = 508 lm). Experiments were performed in single-pulse mode with a test frequency of 0.033 Hz (1 stimulus of 100-ls duration each 30 s). Stimulus intensity was adjusted to produce fEPSPs with amplitudes that corresponded to 40% of maximum. After 30 min of stable baseline recording, LTP was induced by a high-frequency stimulation (HFS) (four 1-s 100 Hz applied at a 20-s interval), using the same stimulus intensity as for baseline recording. After LTP induction, fEPSPs were recorded for 120 min. A Scientifica Slicemaster setup was used to amplify, digitize and record data. fEPSP amplitude measured semi-automatically using ClampFit 10.2 software was chosen as a main parameter describing LTP dynamics. The obtained data were then normalized to the baseline response. MS Excel 2003 and OriginPro 8 software was used for statistical analysis (analysis of variance (ANOVA) and t-test).

LY294002 administration LY294002 (Sigma, St. Louis, Missoury, USA), a specific PI3-K inhibitor, was diluted in dimethyl sulfoxide (DMSO) according to manufacturer protocols and aliquoted. Before the electrophysiological experiments, LY294002/DMSO solution was further diluted in ACSF to reach a final LY294002 concentration of 20 lM.

Transduction efficiency control Biochemical control of viral transduction efficiency was performed using a standard enzyme-linked immunosorbent assay (ELISA) for NGF and BDNF concentrations in rat hippocampi. Rats for biochemical experiments were decapitated 7 days after viral injection. Their brains were removed, frozen in liquid nitrogen, and stored at 20 °C. A Millipore CYT304 ChemiKine NGF Sandwich ELISA kit and a Millipore CYT306 ChemiKine BDNF Sandwich ELISA kit were used according to manufacturer protocols. Briefly, hippocampi were homogenized in icecold buffer and centrifuged. The supernatant was transferred in wells preconjugated with anti-NGF or antiBDNF antibodies respectively, incubated at +4 °C overnight and then washed. Primary and secondary antibodies were added in each well, the plate was incubated at room temperature for 3 h on a shaker and washed three times between incubation with antibodies. Peroxidase reaction was induced by the addition of TBM/E solution and stopped by stop-solution after 15 min. The plate was then immediately read in a Wallac Victor plate-reader at 450 nm and the results

were analyzed using standard calibration curves of NGF and BDNF standards. Western-blot analysis Control hippocampal slices preincubated for 1 h in ACSF or Ab/ACSF were directly lysed in loading buffer and equal amounts of lysates were loaded onto 8% sodium dodecyl sulfate - polyacrylamide gel (SDS-PAAG). After overnight transfer on polyvinylidene difluoride (PVDF)membrane, blocking in 5% milk/(Tris buffered salineTween) TBS-T was performed during 1 h at room temperature. Membranes were stained with anti-TrkB antibodies (SantaCruz, Dallas, Texas, USA, 1:500) followed by anti-rabbit horseradish peroxidase (HRP) secondary antibodies staining. Signal detection was performed with Pierce ECL Detection Reagent kit.

RESULTS Determination of Ab efficient concentration for the blockade of MPP-DG LTP We have previously demonstrated that depression of in vivo LTP caused by i.c.v. injection of Ab (25–35) can be rescued by chronic overexpression of human NGF in the hippocampus (unpublished results). To address the question about specificity of NGF action compared to other neurotrophins and describe the involved signaling cascades, we performed in vitro experiments. Hippocampal slices from P20 to P25 rats were preincubated for 1 h with 20, 50 and 100 nM Ab (25–35) or 50 nM reverse Ab (35–25), respectively. After incubation the slices were transferred to a recording chamber and LTP was induced by HFS of MPP after 30 min of stable baseline recording. As shown on Fig. 1 slices incubated with 20 nM Ab demonstrated potentiation of synaptic transmission similar to non-treated control and to reverse Ab-treated slices (207.1 ± 10.5% n = 44 vs. 200.7 ± 10.8% n = 8 and 196.0 ± 14.3% n = 5; mean ± SE, percent to baseline; t = 1.4 < t Critical = 1.65 p = 0.16 and t = 2.5 > t Critical = 1.65 p = 0.01). In contrast, incubation of slices with 50 nM or 100 nM Ab led to complete abrogation of LTP induction in the MPP-DG synapse (106.1 ± 2.7% n = 5 for 50 nM Ab, 90.2 ± 3.4 n = 5 for 100 nM Ab). Both 50 nM and 100 nM Ab groups differed significantly from either control (t = 31.1 > t Critical = 1.65 p < 0.0001 and t = 36.1 > t Critical = 1.65 p < 0.0001), reverse Ab (t = 29.7 > t Critical = 1.65 p < 0.0001 and t = 34.7 > t Critical = 1.65 p < 0.0001) or 20 nM groups (t = 31.1 > t Critical = 1.65 p < 0.0001 and t = 35.8 > t Critical = 1.65 p < 0.0001). For further experiments 50 nM concentration of Ab (25–35) was chosen. Lentiviral delivery of NGF and BDNF genes led to a significant elevation of neurotrophin’s concentration To investigate how lentiviral delivery of neurotrophin genes affects their tissue concentration we performed ELISA analysis of hippocampal homogenates. For this purpose hippocampi were taken from animals that

A. D. Ivanov et al. / Neuroscience 289 (2015) 114–122

117

Fig. 1. Application of Ab leads to in vitro LTP impairment in the dentate gyrus. Slices were incubated 1 h in 20, 50 and 100 nM Ab (25–35)/ACSF solution before baseline recording and LTP induction by HFS.

received virus injections 7 days prior to tissue collection. Corresponding neurotrophin concentrations were shown to be almost twice higher in the injected hippocampi of the experimental animals, than in contralateral hippocampi or compared to samples from the control virus group. The average NGF concentration after pCMV-NGF injection was 1.72 ± 0.05 pg/mg prot. (n = 6) for ipsi- vs. 0.79 ± 0.008 pg/mg prot. (n = 6) for contralateral hippocampi, and 0.77 ± 0.10 pg/mg prot. (n = 6) for control pCMV-EGFP injection (Fig. 2). No significant differences were found between both ipsiand contralateral pCMV-EGFP groups and contralateral pCMV-NGF group: F(2,15) = 0.39 p = 0.68 (ANOVA). The ipsilatelal pCMV-NGF group, however, differed significantly from both contralateral (t = 9.91 > t Critical = 2.30 p < 0.0001) and pCMV-EGFP control groups (t = 8.83 > t Critical = 2.36 p < 0.0001). The average BDNF concentration after pCMV-BDNF injection was 2.52 ± 0.12 pg/mg prot. (n = 8) for

ispi- vs. 1.29 ± 0.26 pg/mg prot. (n = 8) for contralateral hippocampi, and 1.41 ± 0.07 pg/mg prot. (n = 6) for control pCMV-EGFP injection (Fig. 3). Again, no significant differences were found between both ipsiand contralateral pCMV-EGFP groups and contralateral pCMV-BDNF group: F(2,14) = 3.09 p = 0.07 (ANOVA). The ipsilateral pCMV-BDNF group differed significantly from both contralateral (t = 9.85 > t Critical = 2.30 p < 0.0001) and pCMV-EGFP control groups (t = 8.94 > t Critical = 2.36 p < 0,0001). NGF overexpression protects hippocampal LTP from beta-amyloid-induced impairment To test if lentiviral injection in DG influences electrophysiological properties of neurons per se we used control pCMV-EGFP lentivirus. Neither baseline transmission nor LTP induction was affected in pCMVEGFP-injected animals compared to naı¨ ve controls

Fig. 2. Increase in NGF concentration 1 week after lentiviral transduction. Left column – control group, injection of pCMV virus, second column – contralateral hippocampi of pCMV-injected animals, third column – injection of pCMV-NGF virus, right column – contralateral hippocampi from pCMV-NGF-injected animals.

118

A. D. Ivanov et al. / Neuroscience 289 (2015) 114–122

Fig. 3. Increase in BDNF concentration 1 week after lentiviral transduction. Left column – control group, injection of pCMV virus, second column – contralateral hippocampi of pCMV-injected animals, third column – injection of pCMV-BDNF virus, right column – contralateral hippocampus from pCMV-BDNF-injected animals.

(196.8 ± 12.3% n = 6 vs. 200.7 ± 10.8% n = 8). Moreover, injection of lentiviruses encoding either NGF (195.4 ± 14.7% n = 6) or BDNF (201.3 ± 18.3% n = 5) did not produce detectable changes in basal synaptic transmission or magnitude of LTP: F(3,1200) = 0.90 p = 0.44 (ANOVA). It is worth to note that elevation of BDNF did not lead to changes in LTP dynamics (Fig. 4). We did not record anything similar to the well-known BDNF-LTP phenomena, most likely, because of the stable long-term increase in BDNF concentration, which differs from a single application normally used to provoke BDNF-LTP. To study the protective effect of neurotrophins we incubated slices from pCMV-NGF or pCMV-BDNFinjected animals with 50 nM Ab (25–35) as described in ‘Experimental procedures’ section. After 1 h of incubation slices were transferred to the recording chamber for baseline recording and LTP induction (Fig. 5). As expected, incubation with Ab prevented LTP induction in both naı¨ ve + Ab (106.1 ± 2.7% n = 5) and

pCMV-EGFP + Ab (104.4 ± 4.6% n = 6) groups (t = 3.4 > t Critical = 1.65 p < 0.0001). Interestingly, no LTP was observed in slices obtained from pCMVBDNF-injected animals (98.3 ± 2.9% n = 5), but this was not statistically similar to both Ab (t = 22.0 > t Critical = 1.65 p < 0.0001) and pCMV-EGFP + Ab (t = 15.9 > t Critical = 1.65 p < 0.0001) groups results. In contrast, the NGF group slices still demonstrated normal LTP magnitude and dynamics (200.3 ± 10.1% n = 6). This result was significant when compared to Ab (t = 31.4 > t Critical = 1.65 p < 0.0001), pCMV-EGFP + Ab (t = 31.9 > t Critical = 1.65 p < 0.0001) and pCMV-BDNF + Ab (t = 33.0 > t Critical = 1.65 p < 0.0001) groups. To investigate if the absence of any BDNF protective effect depends on its receptor expression or proteolytic cleavage due to Ab treatment we performed Westernblot analysis on the protein lysates obtained from slices preincubated with Ab/ACSF. We did not detect any difference in the expression levels of TrkB or TrkB

Fig. 4. Effects of NGF and BDNF overexpression on in vitro LTP in normal conditions. fEPSPs representative traces included.

119

A. D. Ivanov et al. / Neuroscience 289 (2015) 114–122

Fig. 5. Effects of NGF and BDNF overexpression on in vitro LTP under Ab-induced impairment. fEPSPs representative traces included.

signaling plays a major role in the observed effect of long-term elevation of NGF concentration.

DISCUSSION

Fig. 6. TrkB receptors Western blotting in Ab-treated and naı¨ ve control slices.

FL/TrkB Tc ratio between control and Ab-treated samples (Fig. 6). Effect of NGF depends on PI3K-signaling cascade The observed effect of NGF can be explained by sustained activation of TrkA-dependent intracellular signaling pathways or structural changes due to elevated expression of target genes. Of the three TrkA-activated kinase cascades the most prominent candidate is the PI3K/Akt cascade, which is related to neuronal survival and synaptic transmission maintenance (Skaper, 2008). To examine its role in the effect of NGF overexpression we used a selective inhibitor of phosphoinositol-3-kinase, LY294002. LY294002 is known to have no impact on LTP induction and maintenance in naı¨ ve acute brain slices (Bruel-Jungerman et al., 2009; Peineau et al., 2007; Mans et al., 2010). We incubated hippocampal slices obtained from pCMV-NGF injected animals for 1 h before baseline recording in LY294002 (Fig. 7). LY294002 did not affect LTP induction if applied alone in both pCMVNGF + LY294002 and control + LY294002 groups (200.5 ± 17.2% n = 4 and 200.0 ± 14.6% n = 5). ANOVA revealed no significant differences between those groups and the naı¨ ve, pCMV-EGFP and pCMVNGF groups: F(4,1500) = 0.62 p = 0.65. When coapplied with Ab, LY294002 abolished the NGF protective effect (106.8 ± 4.4% n = 6; significantly different from the pCMV-NGF + LY294002 group, t = 30.2 > t Critical = 1.65 p < 0.0001). This suggests that PI3K-dependent

Here we investigated the influence of chronic elevation of hNGF or hBDNF concentrations in the DG on in vitro LTP dynamics after acute Ab treatment. Application of betaamyloid (25–35) at a concentration 50 nM completely abolished LTP at the MPP–DG synapses, which is consistent with previous data (Wang et al., 2002; Rowan et al., 2003). At the same time, an increase in NGF but not BDNF concentration completely prevented the observed beta-amyloid-induced LTP impairment. Inhibitory analysis revealed that this effect is dependent on PI3K-signaling. We used the well-known Ab-based model of Alzheimer disease-like pathology, which includes disruption of synaptic plasticity. For elevation of neurotrophin concentration we used lentiviral gene delivery since lentiviruses allow to get stable long-term transgene expression and minimal side-effects such as inflammation (Tuszynski, 2007a,b; Cattaneo et al., 2008; Tukhbatova et al., 2011). As expected, seven days after viral injections we observed an increase in NGF and BDNF concentrations, by up to 220% and 180%, respectively. The operated rats behaved normally and only a thin scar from the syringe needle was seen on the slices. Both NGF and BDNF are essential for neuronal survival and stress-response in various pathological conditions. Lack of NGF trophic support directly leads to neurodegeneration and cell loss (Snider, 1994; Ruberti et al., 2000; Allen and Dawbarn, 2006; Capsoni and Cattaneo, 2006; Skaper, 2008). Pharmacological blockade of NGF processing leads to Alzheimer’s disease-like behavioral alterations and cholinergic neuron degradation in rats (Allard et al., 2012), while NGF injections restore cholinergic neurons phenotype in a mouse model of Down’s syndrome (Cooper et al., 2001). Moreover, NGF injection prevents the degeneration of cholinergic neurons after fornix leisure or administration of toxins (Williams et al., 1986; Koliatsos et al., 1990; Charles et al., 1996; Ruberti et al., 2000; Blesch et al., 2005). Intranasal administration of NGF significantly improved rats’

120

A. D. Ivanov et al. / Neuroscience 289 (2015) 114–122

Fig. 7. Effect on PI3K cascade blockade by LY294002 on LTP dynamics in NGF-overexpressing slices in normal and pathological (Ab) conditions. fEPSPs representative traces included.

performance in the beam walk test and Morris water maze after traumatic brain injury due to downregulation of APP and Ab (1–42) levels (Tian et al., 2012). In our previous work we have demonstrated a protective effect of NGF overexpression on i.c.v. Ab injection caused hippocampal LTP impairment in vivo (Sh. S. Uzakov et al., unpublished results). However in that study we used a less effective lentiviral system with a neuron-specific CaMKII promoter, which led to a smaller increase in NGF concentration and to a partial but not full LTP rescue. BDNF was repeatedly shown to be critically important for LTP induction, consolidation and maintenance (Bramham and Mesaoudi, 2005; Lu et al., 2008; Gomez-Palacio-Schjetnan and Escobar, 2013; Park and Poo, 2013). BNDF-knockouts demonstrate both LTP and basic transmission impairment in the hippocampus, which can be rescued by BDNF application (Korte et al., 1996; Patterson et al., 1996). However, it remains unclear as to whether both NGF and BDNF perform equally in different pathological conditions and their effect on electrophysiological properties of neurons under acute application of pathogen. In this paper we showed that despite a strong similarity in primary structure and activated signaling cascades, the two neurotrophins act differently in our model. While NGF had a profound protective effect against Ab pathological impact, BDNF failed to protect LTP. As the beneficial role of BDNF in various Alzheimer Disease transgenic models is documented in the literature (Nagahara et al., 2009, 2013; Zhang et al., 2013), the observed difference in the effects of NGF and BDNF is due to different patterns of neurotrophin receptor (TrkA and TrkB) activity. It was shown in a recent paper by Jero´nimo-Santos and co-authors that incubation of cortical neurons with amyloid-beta leads to calpainmediated cleavage of TrkB rendering the receptors nonfunctional (Jero´nimo-Santos et al., 2014). To test for this possibility we performed Western blot analysis of protein lysates from Ab-treated and naı¨ ve control slices and observed no difference in the ratio of full length and truncated forms of receptors between these groups. An

alternative explanation of this effect could be abnormalities in retrograde trafficking of BDNF-TrkB complex and its downstream signaling (i.e., Erk5 activation and corresponding gene expression) as shown in (Poon et al., 2013). The effect of Ab-amyloid on synaptic plasticity is complex. It includes activation of p38/MAPK and subsequent internalization of AMPAR, activation of caspases followed by partial proteolytic cleavage of the TrkB receptors (Hsieh et al., 2006; D’Amelio et al., 2011; Nikolaev et al., 2009; Jero´nimo-Santos et al., 2014). Our results suggest that long-term expression of NGF can neutralize these adverse effects of beta-amyloid. To explore the mechanisms underlying the NGF neuroprotective action, we applied a selective PI3K inhibitor LY294002. Preincubation of slices from NGF-injected animals with LY294002 alone did not have any effect on the induction or magnitude of LTP, but combined action of Ab and LY294002 led to complete LTP blockade. This suggests that the activation of PI3K-downstream signaling cascades is critical for the NGF-dependent protective effect. One of the major PI3K cascade components is Akt, a protein kinase with clear neuroprotective potential (Lesne´ et al., 2005; Ma et al., 2009; Zeng et al., 2011). It would be interesting to further perform similar experiments with the constitutively active Akt mutant, to explore Akt’s role in NGF’s neuroprotective action.

CONCLUSIONS The use of neurotrophic factors, primarily NGF and BDNF, as therapeutic agents in cases of different neuropathologies has been widely described in the literature for many years (Aloe et al., 2012 for review). However, most studies on this subject were conducted on cell cultures or on specific transgenic animal models. In our present work we demonstrate with a simple in vitro Ab-based model, that NGF, but not BDNF may act as a neuroprotective and pro-survival agent in the case of Ab-induced acute synaptic plasticity malfunction. We also found that this effect of NGF depends on PI3K/ Akt cascade signaling.

A. D. Ivanov et al. / Neuroscience 289 (2015) 114–122 Acknowledgments—The authors are grateful to Dr. Alon Chen (Weizmann Institute, Rehovot, Israel) for providing the lentiviral packaging plasmids. The authors especially thank Dr. Andrew Irving and Dr. Alexey Bolshakov for manuscript language edition. This study was supported by RFBR (12-04-32121), Presidium of RAS (‘‘Fundamental sciences for medicine’’ and ‘‘Molecular mechanisms of physiological functions’’ Programs) and partially by RSCF (14-25-0072).

REFERENCES Allard S, Leone WC, Pakavathkumar P, Bruno MA, Ribeiro-da-Silva A, Cuello AC (2012) Impact of the NGF maturation and degradation pathway on the cortical cholinergic system phenotype. J Neurosci 32:2002–2012. Allen SJ, Dawbarn D (2006) Clinical relevance of the neurotrophins and their receptors. Clin Sci 110:175–191. Aloe L, Rocco ML, Bianchi P, Manni L (2012) Nerve growth factor: from the early discoveries to the potential clinical use. J Transl Med 10:239. Beck T, Lindholm D, Castren E, Wree A (1994) Brain-derived neurotrophic factor protects against ischemic cell damage in rat hippocampus. J Cereb Blood Flow Metab 14:689–692. Bemelmans AP, Husson I, Jaquet M, Mallet J, Kosofsky BE, Gressens P (2006) Lentiviral-mediated gene transfer of brainderived neurotrophic factor is neuroprotective in a mouse model of neonatal excitotoxic challenge. J Neurosci Res 83:50–60. Blesch A, Conner J, Pfeifer A, Gasmi M, Ramirez A, Britton W, Alfa R, Verma I, Tuszynski MH (2005) Regulated lentiviral NGF gene transfer controls rescue of medial septal cholinergic neurons. Mol Ther 11:916–925. Bramham CL, Mesaoudi E (2005) BDNF function in adult synaptic plasticity: the synaptic consolidation hypothesis. Prog Neurobiol 76:99–125. Bruel-Jungerman E, Veyrac A, Dufour F, Horwood J, Laroche S, Davis S (2009) Inhibition of PI3K-Akt signaling blocks exercisemediated enhancement of adult neurogenesis and synaptic plasticity in the dentate gyrus. PLos One 4:e7901. Capsoni S, Giannotta S, Cattaneo A (2002) Beta-amyloid plaques in a model for sporadic Alzheimer’s disease based on transgenic anti-nerve growth factor antibodies. Mol Cell Neurosci 21:15–28. Capsoni S, Cattaneo A (2006) On the molecular basis linking Nerve Growth Factor (NGF) to Alzheimer’s disease. Cell Mol Neurobiol 26:619–633. Cattaneo A, Capsoni S, Paoletti F (2008) Towards non invasive nerve growth factor therapies for Alzheimer’s disease. J Alzheimers Dis 15:255–283. Chapman PF, Falinska AM, Knevett SG, Ramsay MF (2001) Genes, models and Alzheimer’s disease. Trends Genet 17:254–261. Charles V, Mufson EJ, Friden PM, Bartus RT, Kordower JH (1996) Atrophy of cholinergic basal forebrain neurons following excitotoxic cortical lesions is reversed by intravenous administration of an NGF conjugate. Brain Res 728:193–203. Chen Q-S, Wei W-Zh, Shimahara T, Xie C-W (2002) Alzheimer amyloid b-peptide inhibits the late phase of long-term potentiation through calcineurin-dependent mechanisms in the hippocampal dentate gyrus. Neurobiol Learn Mem 77:354–371. Conner JM, Franks KM, Titterness AK, Russell K, Merrill DA, Christie BR, Sejnowski TJ, Tuszynski MH (2009) NGF is essential for hippocampal plasticity and learning. J Neurosci 29:10883–10889. Cooper JD, Skepper JN, da Penha Berzaghi M, Lindholm D, Sofroniew MV (1996) Delayed death of septal cholinergic neurons after excitotoxic ablation of hippocampal neurons during early postnatal development in the rat. Exp Neurol 139:143–155. Cooper JD, Salehi A, Delacroix J-D, Howe CL, Belichenko PV, ChuaCouzen J, Kilbridge JF, Carlson EJ, Epstein CJ, Mobley WC (2001) Failed retrograde transport of NGF in a mouse model of Down’s syndrome: reversal of cholinergic neurodegenerative

121

phenotypes following NGF infusion. Proc Natl Acad Sci U S A 98:10439–10444. Counts SE, Mufson EJ (2005) The role of Nerve Growth Factor in cholinergic basal forebrain degeneration in prodromal Alzheimer disease. J Neuropathol Exp Neurol 64:263–272. Cullen WK, Suh YH, Anwyl R, Rowan MJ (1997) Block of LTP in rat hippocampus in vivo by b-amyloid precursor protein fragments. NeuroReport 8:3213–3217. D’Amelio M, Cavallucci V, Middei S, Marchetti C, Pacioni S, Ferri A, Diamantini A, De Zio D, Carrara P, Battistini L, Moreno S, Bacci A, Ammassari-Teule M, Marie H, Cecconi F (2011) Caspase-3 triggers early synaptic dysfunction in a mouse model of Alzheimer’s disease. Nat Neurosci 14:69–76. Dewachter I, Reverse´ D, Caluwaerts N, Ris L, Kuipe´ri C, van den Haute C, Spittaels K, Umans L, Serneels L, Thiry E, Moechars D, Mercken M, Godaux E, van Leuven F (2002) Neuronal deficiency of presenilin 1 inhibits amyloid plaque formation and corrects hippocampal long-term potentiation but not a cognitive defect of amyloid precursor protein [V717I] transgenic mice. J Neurosci 22:3445–3453. Freir DB, Holscher C, Herron CE (2001) Blockade of long-term potentiation by beta-amyloid peptides in the CA1 region of the rat hippocampus in vivo. J Neurophysiol 85:708–713. Giacchino J, Criado JR, Games D, Henriksen S (2000) In vivo synaptic transmission in young and aged amyloid precursor protein transgenic mice. Brain Res 876:185–190. Gomez-Palacio-Schjetnan A, Escobar ML (2013) Neurotrophins and synaptic plasticity. Curr Top Behav Neurosci 15:117–136. Houeland G, Romani A, Marchetti C, Amato G, Capsoni S, Cattaneo A, Marie H (2010) Transgenic mice with chronic NGF deprivation and Alzheimer’s disease-like pathology display hippocampal region-specific impairments in short- and long-term plasticities. J Neurosci 30:13089–13094. Hsieh H, Boehm J, Sato C, Iwatsubo T, Tomita T, Sisodia S, Malinow R (2006) AMPAR removal underlies Abeta-induced synaptic depression and dendritic spine loss. Neuron 52:831–843. Huang EJ, Reichardt LF (2001) Neurotrophins: roles in neuronal development and function. Annu Rev Neurosci 24:677–736. Husson I, Rangon CM, Lelie`vre V, Bemelmans AP, Sachs P, Mallet J, Kosofsky BE, Gressens P (2005) BDNF-induced white matter neuroprotection and stage-dependent neuronal survival following a neonatal excitotoxic challenge. Cereb Cortex 15:250–261. Jero´nimo-Santos A, Vaz SH, Parreira S, Rapaz-Le´rias S, Caetano AP, Bue´e-Scherrer V, Castre´n E, Valente CA, Blum D, Sebastia˜o AM, Dio´genes MJ (2014) Dysregulation of TrkB receptors and BDNF function by amyloid-b peptide is mediated by calpain. Cereb Cortex. http://dx.doi.org/10.1093/cercor/bhu105. Koliatsos VE, Nauta HJ, Clatterbuck RE, Holtzman DM, Mobley WC, Price DL (1990) Mouse nerve growth factor prevents degeneration of axotomized basal forebrain cholinergic neurons in the monkey. J Neurosci 10(12):3801–3813. Korte M, Griesbeck O, Gravel C, Carroll P, Staiger V, Thoenen H, Bonhoeffer T (1996) Virus-mediated gene transfer into hippocampal CA1 region restores long-term potentiation in brain-derived neurotrophic factor mutant mice. Proc Natl Acad Sci U S A 93:12547–12552. Larson J, Lynch G, Games D, Seubert P (1999) Alterations in synaptic transmission and long-term potentiation in hippocampal slices from young and aged PDAPP mice. Brain Res 840:23–35. Lesne´ S, Gabriel C, Nelson DA, White E, Mackenzie ET, Vivien D, Buisson A (2005) Akt-dependent expression of NAIP-1 protects neurons against amyloid-{beta} toxicity. J Biol Chem 280:24941–24947. Lu B, Pang PT, Woo NH (2005) The yin and yang of neurotrophin action. Nat Rev Neurosci 6:603–614. Lu Y, Christian K, Lu B (2008) BDNF: a key regulator for proteinsynthesis dependent LTP and long-term memory. Neurobiol Learn Mem 89(3):312–323. Ma R, Xiong N, Huang C, Tang Q, Hu B, Xiang J, Li G (2009) Erythropoietin protects PC12 cells from beta-amyloid (25–35)-

122

A. D. Ivanov et al. / Neuroscience 289 (2015) 114–122

induced apoptosis via PI3K/Akt signaling pathway. Neuropharmacology 56:1027–1034. Mans RA, Chowdhury N, Cao D, McMahov LL, Li L (2010) Simvastatin enhances hippocampal long-term potentiation in C57BL/6 mice. Neuroscience 166:435–444. Mocchetti I, Brown M (2008) Targeting neurotrophin receptors in the central nervous system. CNS Neurol Disord Drug Targets 7:71–82. Moechars D, Dewachter I, Lorent K, Reverse´ D, Baekelandt V, Naidu A, Tesseur I, Spittaels K, Haute CV, Checler F, Godaux E, Cordell B, van Leuven F (1999) Early phenotypic changes in transgenic mice that overexpress different mutants of amyloid precursor protein in brain. J Biol Chem 274:6483–6492. Nagahara AH, Merrill DA, Coppola G, Tsukada S, Schroeder BE, Shaked GM, Wang L, Blesch A, Kim A, Conner JM, Rockenstein E, Chao MV, Koo EH, Geschwind D, Masliah E, Chiba AA, Tuszynski MH (2009) Neuroprotective effects of brain-derived neurotrophic factor in rodent and primate models of Alzheimer’s disease. Nat Med 15(3):331–337. Nagahara AH, Mateling M, Kovacs I, Wang L, Eggert S, Rockenstein E, Koo EH, Masliah E, Tuszynski MH (2013) Early BDNF treatment ameliorates cell loss in the entorhinal cortex of APP transgenic mice. J Neurosci 33(39):15596–15602. Namiki J, Kojima A, Tator CH (2000) Effect of brain-derived neurotrophic factor, nerve growth factor, and neurotrophin-3 on functional recovery and regeneration after spinal cord injury in adult rats. J Neurotrauma 17(12):1219–1231. Nikolaev A, McLaughlin T, O’Leary DD, Tessier-Lavigne M (2009) APP binds DR6 to trigger axon pruning and neuron death via distinct caspases. Nature 457:981–989. Park H, Poo MM (2013) Neurotrophin regulation of neural circuit development and function. Nat Rev Neurosci 14:7–23. Patterson SL, Abel T, Deuel TA, Martin KC, Rose JC, Kandel ER (1996) Recombinant BDNF rescues deficits in basal synaptic transmission and hippocampal LTP in BDNF knockout mice. Neuron 16(6):1137–1145. Paxinos G, Watson C (2005) The Rat Brain in Stereotaxic Coordinates. Elsevier: Academic Press, ISBN 0-12-088472-0. Peineau S, Taghibiglou C, Bradley C, Wong TP, Liu L, Lu J, Lo E, Wu D, Saule E, Bouschet T, Matthews P, Isaac JTR, Bortolotto ZA, Wang YT, Collingridge GL (2007) LTP inhibits LTD in the hippocampus via regulation of GSK3b. Neuron 53:703–717. Poon WW, Carlos AJ, Aguilar BL, Berchtold NC, Kawano CK, Zograbyan V, Yaopruke T, Shelanski M, Cotman CW (2013) bAmyloid (Ab) oligomers impair brain-derived neurotrophic factor retrograde trafficking by down-regulating ubiquitin C-terminal hydrolase, UCH-L1. J Biol Chem 288(23):16937–16948. Reichardt LF (2006) Neurotrophin-regulated signaling pathways. Philos Trans R Soc Lond B Biol Sci 361:1545–1564. Rowan MJ, Klyubin I, Cullen WK, Anwyl R (2003) Synaptic plasticity in animal models of early Alzheimer’s disease. Philos Trans R Soc Lond B Biol Sci 358:821–828. Ruberti F, Capsoni S, Comparini A, Di Daniel E, Franzot J, Gonfloni S, Rossi G, Berardi N, Cattaneo A (2000) Phenotypic knockout of

nerve growth factor in adult transgenic mice reveals severe deficits in basal forebrain cholinergic neurons, cell death in the spleen, and skeletal muscle dystrophy. J Neurosci 20(7):2589–2601. Scha¨bitz WR, Sommer C, Zoder W, Kiessling M, Schwaninger M, Schwab S (2000) Intravenous brain-derived neurotrophic factor reduces infarct size and counter regulates Bax and Bcl-2 expression after temporary focal cerebral ischemia. Stroke 31(9):2212–2217. Skaper SD (2008) The biology of neurotrophins, signaling pathways, and functional peptide mimetics of neurotrophins and their receptors. CNS Neurol Disord Drug Targets 7:46–62. Snider WD (1994) Functions of the neurotrophins during nervous system development: what the knockouts are teaching us. Cell 77(5):627–638. Thal LJ (1996) Neurotrophic factors. Prog Brain Res 106:327–330. Tian L, Guo R, Yue X, Lu Q, Ye X, Wang Zh, Chen Zh, Wu B, Xu G, Liu X (2012) Intranasal administration of nerve growth factor ameliorates b-amyloid deposition after traumatic brain injury in rats. Brain Res 1440:47–55. Tukhbatova GR, Kuleshova EP, Stepanichev MYu, Ivanov AD, Salozhin SV (2011) Optimisation of a preparation of lentiviral particles for transduction of neurons in vivo. Neurochem J 5(4):294–300. Tuszynski MH, U HS, Alksne J, Bakay RA, Pay MM, Merrill D, Thal LJ (2002) Growth factor gene therapy for Alzheimer disease. Neurosurg Focus 13(5):e5. Tuszynski MH (2007a) Nerve growth factor gene therapy in Alzheimer disease. Alzheimer Dis Assoc Disord 21(2):179–189. Tuszynski MH (2007b) Nerve growth factor gene delivery: animal models to clinical trials. Dev Neurobiol 67(9):1204–1215. Volosin M, Song W, Almeida RD, Kaplan DR, Hempstead BL, Friedman WJ (2006) Interaction of survival and death signaling in basal forebrain neurons: roles of neurotrophins and proneurotrophins. J Neurosci 26(29):7756–7766. Wang HW, Pasternak JF, Kuo H, Ristic H, Lambert MP, Chromy B, Viola KL, Klein WL, Stine WB, Krafft GA, Trommer BL (2002) Soluble oligomers of beta amyloid (1–42) inhibit long-term potentiation but not long-term depression in rat dentate gyrus. Brain Res 924:133–140. Williams LR, Varon S, Peterson GM, Wictorin K, Fischer W, Bjorklund A, Gage FH (1986) Continuous infusion of nerve growth factor prevents basal forebrain neuronal death after fimbria fornix transaction. Proc Natl Acad Sci U S A 83(23):9231–9235. Zeng KW, Wang XM, Ko H, Kwon HC, Cha JW, Yang HO (2011) Hyperoside protects primary rat cortical neurons from neurotoxicity induced by amyloid b-protein via the PI3K/Akt/Bad/ Bcl(XL)-regulated mitochondrial apoptotic pathway. Eur J Pharmacol 672:45–55. Zhang W, Wang PJ, Li MH, Gao XL, Gu GJ, Shao ZH (2013) 1HMRS can monitor metabolites changes of lateral intraventricular BDNF infusion into a mouse model of Alzheimer’s disease in vivo. Neuroscience 245:40–49.

(Accepted 23 December 2014) (Available online 13 January 2015)