Background
The progressive, permanent loss of neurons in Alzheimer’s disease (AD) means the potential for effective intervention declines as the disease advances [
1]. Examining AD pathology at a late stage also makes identifying the primary drivers of the disease cascade challenging, as pivotal processes are masked by a melee of downstream consequences [
2]. Taken together, it is clear that for effective therapeutics to be developed, we must observe, and target, the disease in its initiating phase. Synaptic dysfunction has long been noted as one of the earliest hallmarks of AD [
2], occurring in advance of neuronal death [
3,
4] and correlating best with soluble, rather than plaque-forming, Aβ [
5]. Within as little as 2 years of clinical onset, cortical synapse density can be reduced by 35 % in patients [
6], such loss of synapses being the best correlate of clinical outcome [
7]. Presynaptic terminals seem especially vulnerable at this stage; synaptophysin [
8], rab3a [
9,
10], and synaptobrevin [
10] are all significantly depleted in early AD brain tissue [
11]. Despite other limitations, familial AD (FAD) mouse models recapitulate the synaptic alterations of the early stages of AD. As in human patients, there is a plaque-independent loss of synaptophysin-immunoreactive presynaptic boutons [
12‐
14] as well as disruption of essential presynaptic components within early dystrophic axons [
15]. Direct readouts of synaptic function also reveal deficits; LTP showing more rapid decay in FAD mice [
16]. Such alterations occur in models lacking tau pathology, demonstrating alterations in Aβ processing, at least in the presence of wild-type murine tau [
17], are sufficient to cause some synaptic deficits.
Investigation of the relationship between Aβ and early synaptic deficits would be facilitated by a model system amenable to repeated sampling, imaging and manipulation. While outer cortical layers of FAD mouse brain can be imaged using a cranial window [
18,
19], key areas of pathology such as the hippocampus cannot. Instead, a major, terminal operation is needed to reach them, limiting observations to a single occasion [
20]. Delivery of drugs is also limited by the blood brain barrier or by the acute and invasive nature of intracerebroventricular delivery. In vitro models, particularly of primary cortical neurons, bypass many of these issues, but fail to account for the complexity of the mammalian brain, its specialised neuronal circuits and variety of interacting cell types. Organotypic Hippocampal Slice Cultures (OHSCs) fill an important gap between these two systems. Whilst maintaining a complete hippocampal circuit and associated cell populations for several weeks, they allow for a level of experimental manipulation and accessibility that is unobtainable in vivo [
21]. Screening for potential disease-altering compounds is made easier by the lack of blood-brain barrier or any systemic effects, and the ability to completely alter the extracellular environment in a matter of seconds. By adding (or removing) test compounds at different time points, their ability to prevent, halt or reverse pathology can be easily assessed. As such, OHSCs present an ideal platform to both observe and manipulate the amyloid cascade.
OHSCs from FAD mice have been surprisingly underused in the AD research field, especially to study progressive pathology caused by endogenously produced Aβ. Some studies have focused on applying synthetic Aβ (in various aggregation states) to acute cultures from non-transgenic animals. Such work has highlighted the importance of oligomeric Aβ in AD pathology, due to its enhanced ability to suppress of LTP [
22], cause neuronal death [
23] and alter dendritic spine density [
24]. Other work has focussed on understanding plaque formation and dissolution, showing, for example, that microglia from adult FAD mice (when compared to juveniles or WT controls) are ineffective at clearing synthetic Aβ fibrils [
25], and that neprilysin, insulysin and matrix metalloproteinases can aid clearance of plaques already present in slices prepared from adults [
26]. Occasional studies using OHSCs from FAD mice have used a single timepoint to focus on dendritic spine or branching alterations following transduction with human tau [
27,
28] or manipulation of tau pathology through pharmacological means [
29]. There is also little known about presynaptic pathology in OHSCs, despite reports of extensive axonal swelling and synaptophysin depletion in FAD mice in vivo [
15,
30] and in patients [
31].
Here we describe an OHSC model from TgCRND8 mice [
32] as a tool to explore the early and progressive effects of Aβ in central nervous system (CNS) tissue. We used this strain because amyloid pathology and axonal swelling begin within two months in vivo [
15]. This is broadly within the timeframe for which OHSCs can be maintained [
33], so we hypothesised that early stages of pathology caused by endogenous APP processing products should be detectable. We show that the transgenic slices are viable for in excess of 8 weeks, retain all major CNS cell types and rapidly release soluble Aβ into the culture medium, the production rate of which can be manipulated pharmacologically. While there is no plaque pathology, there is a striking accumulation of intraneuronal Aβ (at least some of it axonal), far more than in vivo, accompanied by a specific and progressive decline in presynaptic proteins. This is now an excellent experimental model for understanding the mechanism of progressive Aβ-induced synapse dysfunction and how it can be prevented.
Methods
Mice
TgCRND8 mice [
32], overexpressing human APP with Swedish (K670N/M671L) and Indiana (V717F) FAD mutations, were maintained as heterozygotes on a 62.5:37.5 sv129: C57BL/6 background, generating transgenic and wild-type (WT) littermate controls. Thy1-mitoCFP [
34] mice were also maintained as heterozygotes on a C57BL/6 background and females were crossed with TgCRND8 males to generate double transgenic mice. Animals were kept on a 12:12hs light: dark cycle at a constant temperature of 19 °C in a pathogen-free environment. All animal work was approved by the Babraham Institute Animal Welfare and Ethical Review Body and UK Home Office, and carried out in accordance with the Animals (Scientific Procedures) Act, 1986, under Project Licence 70/7620.
Organotypic slice cultures
Organotypic cultures of the hippocampus and surrounding cortex were taken from humanely sacrificed P6-P9 mouse pups of either sex according to the method described by de Simoni et al. [
33]. Briefly, brains were rapidly removed and kept in dissection buffer (EBSS+ 25 mM HEPES+ 1 X Penicillin/Streptomycin) on ice. From this point, until plating, all equipment and tissue was kept ice cold. Brains were bisected at the midline then the cut sides glued (Loctite), face down onto a vibratome stage and flooded with dissection media. 350 μm sagittal slices (6 per brain) were taken using a Leica VT1000S Vibratome; the hippocampus with surrounding cortex was dissected out using sterile syringe needles whilst the slice was lying on the vibratome blade. The dissected slices were then transferred (using a sterile 3 ml plastic pipette- modified to widen the opening) to Falcon tubes full of ice-cold dissection medium and stored until plating. To plate, slices were transferred (3 slices from the same brain per dish) onto sterile 0.4 μm pore membranes (Millipore PICM0RG50) in 35 mm culture dishes (Nunc). Inserts were kept in 1 ml of maintenance medium (50 % MEM with Glutamax-1 (Life Tech:42360-024), 25 % Heat-inactivated horse serum (Life Tech: 26050-070 ), 23 % EBSS (Life Tech: 24010-043), 0.65 % D-Glucose (Sigma:G8270) , 2 % Penicillin-Streptomycin (Life Tech: 15140-122)and 6 units/ml Nystatin (Sigma: N1638) )and cultures were maintained in incubators at 37 °C, 5 % CO
2 for up to 12 weeks. Two 100 % medium exchanges occurred (5 hs after plating and 4
div) and a 50 % media exchange occurred each week thereafter.
Aβ ELISA and drug treatments
To determine levels of human Aβ1-40 or Aβ1-42 in the slice culture medium, samples were analysed using commercially available ELISA kits (Life Tech: KHB3441/KHB3481). Briefly, culture medium was diluted to bring the expected concentration within the range of the standard curve before being incubated with Aβ detection antibody for 3 hs at room temperature. After washing, samples were incubated with HRP-conjugated anti-rabbit antibody for 30mins, washed, and then incubated with stabilised chromogen for 30mins. The reaction was stopped using an acid-based stop solution and absorbance read at 450 nm using a PheraStar FS plate reader. Samples were run with a standard curve (4-parameter fit) to obtain a concentration readout in pg/ml.
For quantifying Aβ within the slice tissue, material from a single culture membrane (3 slices) was homogenised in 10 μL 5 M Guanidine Hydrochloride supplemented with 1x Protease Inhibitor Cocktail (Roche) for 3-4 hs at room temperature (RT). The sample was then frozen at -20 °C until use. Prior to running in the ELISA, the homogenate was diluted 1:50 in ice cold reaction buffer (Dulbecco’s PBS + 0.03 % Tween + 5 % BSA supplemented with 1x Protease Inhibitor Cocktail) and centrifuged for 20mins at 4 °C at 16,000 x g. The supernatant was then diluted before undergoing ELISA readout as for the media samples. Readout is given as pg of Aβ per slice.
To determine how drug treatment influenced Aβ accumulation, slices were moved to fresh maintenance media for 24 hs. 50 μl of conditioned culture medium was then taken and frozen at -20 °C to act as baseline production readout. Calpain Inhibitor 1 (Sigma: A6185)) or DMSO control was then applied to the culture, with 50 μl of the treated medium dropped onto the slices to ensure complete drug infusion. The medium on top rapidly soaks through the slice, so the oxygen exchange is not hindered during this time. 50 μl aliquots of culture medium are then taken every 24 hs to monitor Aβ production, with readouts normalised to the original 24 h baseline. All drug experiments were run in triplicate, with 3 independent membranes from different mice used per experiment.
Immunofluorescence staining
Membranes were transferred into 6 well plates and slices were fixed for 20mins in 4 % paraformaldehyde in 0.1 M PBS (applied both above and below the membrane insert). To reduce the volumes required for subsequent steps, the membranes were cut free of the plastic inserts and the sections of membrane containing the slices were transferred, using forceps, to individual wells in a 24 well plate. Slices were washed twice in TBS, blocked for 1 h in blocking solution (TBS with 0.5 % Triton X-100 and 3 % Goat Serum) then incubated in 200 μl primary antibody diluted in blocking solution overnight at 4 °C with shaking. Slices were washed 3 times in TBS before being incubated (2 hs, RT in the dark) with Alexa488, 568 or 647 conjugated secondary antibodies (Life Technologies-diluted 1:250 in blocking solution). After a final 3 TBS washes, some slices were counterstained with Thioflavin S, BTA-1, Nissl or Hoechst. Images were captured using a Nikon Confocal Microscope. Primary antibodies used: mouse Tuj1 (Covance 1:1000), rabbit Tuj1 (Sigma 1:500), chicken Tuj1 (Abcam:1:1000) rabbit NFL (Millipore 1:250), mouse MOAB2 (pan specific to Aβ- Millipore 1:1000), rabbit GFAP (Abcam 1:1000), mouse synaptophysin (Dako 1:1000), rabbit PSD95 (Abcam 1:500), rabbit tau (Dako 1:1000), rabbit Iba1 (Wako 1:500) rabbit calbindin and rabbit parvalbumin (Kind gifts from Dr P Emson 1:1000).
Quantification of Aβ positive swellings
To quantify the degree of Aβ positive swellings in OHSCs of different ages, x20 images of MOAB2 staining in the CA1 region were captured (blinded to culture age and using identical microscope settings for each image) then processed using Fijix64 image analysis software [
35]. The MOAB2 (red) channel was isolated and the image threshold manually adjusted to remove background (thresholding was performed blind to culture age, with the original image open in parallel, to ensure the thresholded image accurately represented visible staining). The plugin “despeckle” was applied to remove isolated pixel noise before the “Analyze particles” plugin was run. “Total particle count” results were compared between slices of different ages.
Synaptic marker image capture, processing and puncta quantification
5-week old TgCRND8 and WT slices were imaged according to the synapse quantification protocol adapted from Ippolito and Eroglu [
36‐
38]. Briefly, slices were stained for PSD95 (secondary labelled Alexa-568) and Synaptophysin (secondary labelled Alexa-488) according to the standard immunofluorescence protocol described above. Using a Zeiss 780 confocal x63 oil-immersion objective, image stacks from the CA1 (location of Aβ- positive axonal swellings in TgCRND8 cultures) and CA3 (largely swelling-free) fields were collected. For each slice, the chosen field was imaged using serial optical sections at 0.33 μm for a total of 15 sections (total depth of 5 μm). Maximal intensity projections (MIPs) were generated from 3 consecutive optical sections, resulting in 5 images each displaying 1 μm depth per field section in a slice. Quantification was performed using an imageJ 1.29 plugin [
36,
39,
40] (available from c.eroglu@cellbio.duke.edu). Briefly, 33 μm x 33 μm regions of interest were randomly selected from each MIP and the “Puncta Analyzer” plugin run. Red (PSD95) and green (Synaptophysin) channels were manually thresholded to highlight visible puncta without the introduction of background noise. The plugin provides quantitative data for puncta number in each channel, as well as the number of colocalised puncta. 8 membranes per genotype (from different mice) were analysed, the MIP values from individual slices on the same membrane averaged to give an overall “membrane average”. Throughout image collection and analysis, the experimenter was blind to slice genotype.
Western blotting
Slices were scraped off the membrane, treated with 2x Laemelli buffer + 10 % 2-mercaptethanol (250 μL per 3 slices) vortexed, boiled, then frozen at -20 °C until use. For use, most samples were further diluted 1:2 and loaded 8 μl per lane in a precast 4-20 % gradient gel (Bio-rad). To detect the weaker signal for PSD95, 15 μl of undiluted sample was loaded. After incubation in primary antibody overnight, blots were probed with 1:5000 mouse-700 (Life Technologies) and rabbit-800 (LI-COR) secondary antibodies then imaged using a LI-COR Odyssey detection system. Band intensity (IKK) was quantified using Odyssey software then normalised to Tuj1 signal. Primary antibodies used: mouse synaptophysin (Abcam: 1:1000), rabbit tuj1 (Sigma: 1:2500), rabbit PSD95 (Abcam: 1:500), mouse VAMP2 (Synaptic Systems: 1:10,000) and mouse RT97 (Kind gift from Dr Diane Hanger 1:500). Tuj1-normalised protein expression was then compared between WT and TgCRND8 cultures, TgCRND8 values expressed as a percentage of the WT average.
Statistical analysis
Analysis was conducted using GraphPad Prism software. To assess calpain inhibitor dose response and Aβ swelling count data, one way ANOVAs with Dunnett or Tukey post hoc tests (respectively) were used. For synaptic protein western blot and calpain inhibitor treatment effects, two way ANOVAs with Sidak post hoc tests were conducted. For synaptic puncta counts, Student’s t-tests were used. Results are expressed as mean +/- standard error.
Discussion
Here we describe how long term OHSCs from TgCRND8 mice can be used as a model of Aβ pathology, revealing novel aspects of the disease mechanism that are not easily studied in vivo. We can now explore, in a system highly amenable to manipulation and analysis: release of Aβ, loss of presynaptic proteins, loss of SYP/PSD95 colocalisation and the accumulation of intraneuronal Aβ. We have also seen that addition of calpain inhibitor 1 enhances Aβ production in this model, potentially opening doors to examine how pathological outcomes change with rising Aβ. Whilst the nature and time course of pathology shows some interesting differences from that seen in vivo, long term OHSCs from TgCRND8 mice represent an exciting new tool for research into the early consequences of progressive accumulation of Aβ and other consequences of APP processing.
Loss of presynaptic proteins and puncta correlates spatially and temporally with the appearance of intraneuronal Aβ
Loss of presynaptic proteins is thought to be one of the earliest (and most clinically relevant) changes in human AD [
2,
51] with some reports indicating it precedes extensive postsynaptic changes [
52]. In TgCRND8 OHSCs, the presynaptic proteins synaptophysin and VAMP2 are depleted when compared to WT controls. This corresponds to a region-specific loss of presynaptic puncta, consistent with loss of synapses. Our finding that PSD-95 protein and puncta are not depleted supports the notion that presynaptic changes can precede other alterations [
52‐
54]. The preservation of axon-specific phosphorylated neurofilament also supports localised changes in the immediate presynaptic compartment, and not a wider loss of axons at this stage.
A key future direction will be to determine the cause of the presynaptic changes in the TgCRND8 slices. As plaques do not develop during the experimental timecourse (see below), they are not needed to drive synaptic alterations. Indeed, the lack of tau pathology in TgCRND8 mice also rules out neurofibrillary tangles as a necessary cause of the presynaptic protein depletion we see. Whilst the observed synapse effects are clearly dependent on the huAPP transgene, it is important to remember that OHSCs undergo a period of synaptic reorganisation in culture following the significant tissue injury at the time of slice generation that does not occur in vivo. In WT rat OHSCs, it has been shown that within the first 2 weeks in vitro, the maximal evoked EPSP increases, plateauing between 10 and 15 days in vitro, likely representing an increase in synaptic connections [
55]. APP overexpression has been shown to have a negative effect on synapse development in vitro [
56,
57] so it is possible that the TgCRND8 slices have a reduced capacity for this repair that at least partly underlies the lower presynaptic protein levels.
A clear difference between the WT and TgCRND8 OHSCs that associates both temporally and spatially with the synaptic changes is the gradual appearance of Aβ-containing swellings, most commonly located in the alveus adjacent to CA1. There is an increase in the presence of such structures between 2 and 5 weeks in vitro, which closely parallels the development of presynaptic protein deficiency. The loss of synaptic structures, as measured by PSD95/SYP puncta colocalisation, also spatially correlates with the regions affected by Aβ positive swellings. Previous studies have shown that intraneuronal Aβ accumulation in synaptic compartments is directly associated with abnormal cellular morphology [
58] whilst another study found that reducing the intraneuronal pool of Aβ through synaptic activation was protective against loss of synaptic proteins [
59]. The role of intraneuronal Aβ in pathogenesis has been a controversial topic [
43,
60], so the experimental system we report, in which Aβ generation and its release through synaptic activity can be readily manipulated, will be useful in resolving its importance in the disease process.
Intraneuronal Aβ in axonal swellings
Whilst many FAD mouse models have been reported to develop pre-plaque intraneuronal Aβ pathology, [
60‐
63] it is particularly interesting that the appearance of such striking Aβ positive swellings in TgCRND8 OHSCs differs from the corresponding in vivo phenotype in these animals. Whilst adult TgCRND8 mice usually show APP immunoreactivity within dystrophic axons [
15] and there is some evidence for lysosomal Aβ accumulation in older mice [
64], extensive intra-axonal accumulations such as the ones found in the OHSCs are not seen at any age studied in this strain [
15] (Additional file
1: Figure S1). Interestingly, a similar phenomenon has been reported in whole brain slices from adult APP_SDI mice; a failure to develop plaques, but appearance of atypical intraneuronal Aβ staining [
26]. This suggests that a hippocampus in slice culture is more prone to retain Aβ within cells than in vivo so it will be interesting to investigate the mechanism that underlies this difference. The appearance of swellings in WT cultures (albeit without Aβ accumulation) could indicate this is a result of axonal damage during the slice procedure, or another consequence of the slice culture system. Similarly, intraneuronal Aβ accumulation is commonly observed in axonal injury after brain trauma. Studies in humans [
65], pigs [
66] and rats [
67] have all reported that injured, swollen or broken axons can act as sites of Aβ accumulation- particularly in axonal end bulbs. The nature of the OHSC is such that axotomy of certain populations of neurons is unavoidable. Whilst there is evidence for re-organisation and recovery in this model [
68], it may be that in the TgCRND8 cultures (which will already have elevated levels of both APP and Aβ) this injury further seeds accumulation of Aβ. However, as it takes over 3 weeks for the intraneuronal Aβ to accumulate, any link to the initial injury appears likely to involve additional steps.
A further possibility is, due to lack of sequestration in plaques, the OHSCs may be bathed in relatively high concentrations of
soluble Aβ when compared to adult brain. There is evidence to suggest that neurons can actively uptake Aβ, with the axon being highlighted as a potential point of entry [
69]. Indeed, application of synthetic Aβ to OHSCs from WT rats resulted in intraneuronal accumulation of this peptide in CA1 [
70], the same region that is heavily affected in our TgCRND8 OHSCs. Perhaps this region is rendered more vulnerable during the slice procedure resulting in enhanced uptake of exogenous Aβ than would be seen in vivo.
Whatever the cause of the intraneuronal Aβ accumulations in OHSCs, the fact that it differs from the in vivo phenotype will help us to understand the factors that govern the balance between intracellular and extracellular amyloid pathology. By studying the balance between Aβ in the tissue and the medium it should also be possible to investigate factors that influence its rate of extrusion. As both aspects of pathology are present in sporadic human AD [
58,
71,
72] understanding the mechanisms of each could assist in developing effective therapeutic interventions.
TgCRND8 OHSCs do not develop plaques
An unexpected finding in TgCRND8 OHSCs is that plaques fail to develop even after 12 weeks in vitro, over 4 weeks after such pathology would develop in vivo [
15,
32]. A potential explanation for this is that the large volumes of culture medium relative to the small quantity of slice tissue washes Aβ from the slices more effectively than vascular perfusion, preventing the seeding of plaques. Alternatively, it could be that the microglia in the OHSCs are more effective at preventing plaque formation, perhaps as a result of activation from the initial slice preparation. A recent paper demonstrated that microglia from juvenile 5xFAD mice or WT controls are highly effective at clearing synthetic Aβ fibrils applied to WT OHSCs, whilst adult 5xFAD microglia cannot prevent the formation of aggregates [
25]. It may be that in the long term OHSCs, a more juvenile microglial phenotype is maintained, thus preventing plaque deposition. Understanding exactly how the slice system differs to in vivo will, once again, assist in unpicking the mechanisms behind AD pathology.
Additional uses of the model
The OHSC model we describe has many potential uses in studying the mechanism of amyloid pathology and developing therapeutics, but also a number of limitations. Slice preparation involves massive tissue damage, resulting in axotomy, cell death and activation of inflammatory cells. Plaque pathology does not develop, and there is increased intra-axonal Aβ beyond that seen in vivo. There is also a gradual over proliferation of non-neuronal cells, no vascular system and no electrical input from other brain regions. It should also be remembered that the slices are generated from juvenile mice, where many cells have yet to reach their mature phenotypes.
However, the progressive changes we report in long term TgCRND8 OHSCs have significant potential to be of use to the AD research field. For example, the rapid production of soluble Aβ species from these slices could be utilised as a source of pathogenic amyloid peptides without presupposing which of them is/are the most toxic. These could then be introduced exogenously into other experimental systems such as primary neuronal cultures, or OHSCs of a different genotype, avoiding the use of supraphysiological concentrations of synthetic peptides. This system should also enable studies examining the spread of pathological proteins, and screening, at least at a secondary stage, for compounds that will block Aβ generation or release, or block synaptophysin depletion.
The ability to repeatedly image, or live image, OHSCs is also important. Unlike the in vivo hippocampus, which is difficult to image even using multiphoton microscopy, we have been able to observe live cells such as microglia (stained using isolectin B4 conjugated to Alexa fluora568 (Life Technologies)) (Additional file
2: Figure S2a) and the axonal transport of mitochondria in OHSCs expressing mito-CFP (Additional file
3). We find that axons from the dentate gyrus are easily located with this genetic label (Additional file
2: Figure S2b-c) and repeat imaging of the same slices is possible. Mitochondrial or other axonal transport dynamics have been implicated many times in the pathogenesis of AD. Further work will be needed to develop this transport imaging for quantitative assessment, but probing for differences in the TgCRND8 slices has the potential to enhance understanding in a way that would not be possible in vivo.
Conclusions
In summary, we report the first characterisation of progressive deficiencies in OHSCs from a huAPP mouse model. This reveals both similarities and differences from observations made in the same mouse strain in vivo, thus validating this system as a model for some aspects of pathogenesis. The model will be particularly useful for understanding disease mechanism, both because it can be readily manipulated, repeatedly sampled and imaged, and because the observed differences from in vivo pathology provides a basis for understanding why these occur (for example, the shift from plaques to intraneuronal Aβ). This experimental system also has important potential as a drug-screening platform. Here, candidate drugs can be readily delivered and monitored, and their effects on all relevant cell types and neuronal circuits observed, thus filling a vital gap between primary culture and in vivo studies. We expect this OHSC model will find many applications in AD research.
Abbreviations
AD, Alzheimer’s Disease; Aβ, Amyloid-beta; CNS, Central Nervous System; MIP, Maximum Intensity Projection; OHSC, Organotypic hippocampal slice culture; PSD95, Post synaptic density 95; SYP, Synaptophysin; VAMP2, Vesicle-associated membrane protein 2; WT, Wild-type
Acknowledgements
This work was supported by Alzheimer’s Research UK studentship ARUK-PhD2013-13. We would like to thank Simon Walker and Hanneke Okkenhaug for imaging advice, Anna De Simoni for technical advice, Anne Segonds-Pichon for assistance with statistical analysis, Simon Andrews for assistance with image J plugins for counting Aβ positive swellings and Prof. Eroglu for providing the synaptic puncta image J plugin. Thanks also to Robert Adalbert for his neuroanatomical advice.