Neuroinflammation impairs adaptive structural plasticity of dendritic spines in a preclinical model of Alzheimer’s disease
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To successfully treat Alzheimer’s disease (AD), pathophysiological events in preclinical stages need to be identified. Preclinical AD refers to the stages that exhibit amyloid deposition in the brain but have normal cognitive function, which are replicated in young adult APPswe/PS1deltaE9 (deltaE9) mice. By long-term in vivo two-photon microscopy, we demonstrate impaired adaptive spine plasticity in these transgenic mice illustrated by their failure to increase dendritic spine density and form novel neural connections when housed in enriched environment (EE). Decrease of amyloid plaques by reducing BACE1 activity restores the gain of spine density upon EE in deltaE9 mice, but not the remodeling of neural networks. On the other hand, anti-inflammatory treatment with pioglitazone or interleukin 1 receptor antagonist in deltaE9 mice successfully rescues the impairments in increasing spine density and remodeling of neural networks during EE. Our data suggest that neuroinflammation disrupts experience-dependent structural plasticity of dendritic spines in preclinical stages of AD.
KeywordsPreclinical AD APPswe/PS1deltaE9 mice Dendritic spines Structural plasticity Neuroinflammation
As the most prevalent cause of dementia, Alzheimer’s disease (AD) is characterized by progressive cognitive deficits, amyloid plaques, neurofibrillary tangles (NFTs) and neuronal loss, yet it still lacks an effective cure at the present time [21, 40]. The failure to develop successful pharmacotherapy may, at least partially, be ascribed to the long pathophysiological process, which starts many years before the stage of symptomatic AD . Therefore, much earlier intervention in the asymptomatic or preclinical stages may be required to successfully treat AD [35, 45].
Preclinical AD has been recently defined as the stages when amyloid is being deposited in the brain, but before the onset of any cognitive impairment . Subjects in the preclinical stages are at risk for future cognitive decline . Indeed, the lag between the appearance of amyloid plaques and detectable impairment in cognition is more than a decade [37, 44]. Growing evidence supports the notion that amyloid deposition disrupts functional networks in the brain of cognitively normal elderly [17, 32, 42, 46]. To have a better chance of curing AD, it is therefore crucial to identify pathophysiological events occurring in preclinical stages that precede dementia during the initial formation of amyloid deposits.
Transgenic mouse models are essential research tools for uncovering AD pathogenesis as well as validating new therapeutic approaches. To recapitulate AD pathology, transgenic mouse models carry familial AD gene mutations in the amyloid precursor protein (APP) and/or presenilins (PS) based on the amyloid hypothesis, which postulates an increased production or decreased removal of the APP proteolytic fragment, amyloid β-protein (Aβ), as the primary cause of AD . The transgenes with APP/PS mutations in mouse models lead to the formation of amyloid plaques and subsequent memory loss, but without the development of NFTs and massive neuronal loss . Although these mice fail to replicate all aspects of the disease, they seem to faithfully imitate pre-dementia stages of AD .
Among the APP transgenic mouse models, APPswe/PS1deltaE9 (deltaE9) mice have been widely used. They express APP with the Swedish mutation together with mutant human PS1 with a deletion of exon 9 [23, 39]. Interestingly, in deltaE9 mice, amyloid deposition precedes typical cognitive impairments [22, 50]. Amyloid plaques start to emerge at the age of 4–5 months [4, 14], while the performance of 7-months-old deltaE9 mice is normal in most cognitive tests [27, 34, 50]. The temporal lag between the emergence of amyloid plaques and the onset of dementia consequently provides a critical period to study pathophysiological events related to preclinical AD.
In this study, we used long-term in vivo two-photon microscopy to elucidate the adaptive spine plasticity of deltaE9 mice at young adult age. Our data demonstrated that deltaE9 mice failed to increase spine density and establish novel neural connections when exposed to enriched environment (EE), which was caused by neuroinflammation.
Materials and methods
APPswe/PS1deltaE9 (deltaE9) mice  (Jackson Laboratory) were crossed with GPF-M mice  (Jackson Laboratory) to obtain double transgenic offspring, which were heterozygous for the corresponding genes (deltaE9 +/− × GFP +/−). GFP positive littermates without APP/PS1 transgenes were used as controls (deltaE9 −/− × GFP +/−). BACE1 knockout mice  were also purchased from Jackson Laboratory, and deltaE9 +/− × Bace1 +/− × GFP +/− (deltaE9/Bace +/−) were generated by interbreeding. All transgenic mice were maintained on C57BL/6 background. Female mice at the age of 4–5 months were used. Mice were housed and bred in pathogen-free environment in the animal facility at the Centre for Neuropathology and Prion Research of the Ludwig-Maximilians-University Munich (LMU), with food and water provided ad libidum (21 ± 1 °C, at 12/12 h light/dark cycle). All mice were housed either singly in standard cages (30 × 15 × 20 cm) or in groups in environmentally enriched (EE) cages (80 × 50 × 40 cm) equipped with platforms and variety of toys, which were relocated 3 times per week. Pioglitazone (350 ppm, Actos™) was supplemented into rodent chow. All protocols and procedures involving animals were approved and conducted in accordance with the regulations of LMU and the government of Upper Bavaria (Az. 55.2-1-54-2532-62-12).
Cranial window implantation and in vivo two-photon imaging
The detailed surgical procedure of cranial window implantation has been described previously [13, 20]. In brief, mice were anesthetized by intraperitoneal injection of ketamine/xylazine (120 and 10 mg/kg, respectively). Subsequently, dexamethasone (6 mg/kg) was injected to prevent development of cerebral edema. A piece of skull above the somatosensory cortex was then removed carefully with a dental drill. The exposed brain was cleaned with sterile saline and covered with a round glass coverslip (D = 4 mm). The margin between the glass and skull was sealed with dental cement. Post-surgical mice were subcutaneously injected with carprofen (Pfizer, 4 mg/kg) and cefotaxime (Pharmore, 250 mg/kg). Lentiviruses (LV) encoding IL-1 RA (LV vector was a gift from Dr. van Dam ) were intraparenchymally injected into the cortex before implanting the coverslip when specified. LV encoding GFP was used as a control for evaluating the efficiency of IL-1 RA expression. The injection of the LV (200 nl per time at a titer of ~108 infecting units per ml) was performed at 4 different sites in the exposed area of brain at the depth of 700–800 µm. After 4 weeks of recovery period, mice were imaged by using a LSM 7MP microscope (Zeiss) equipped with a 20× objective (NA 1.0; Zeiss). Mice were anesthetized with isoflurane (1 % in 95 % O2 and 5 % CO2) and placed on a heating pad to keep the body temperature at 37 °C. Apical dendrites originating from GFP-labeled layer V pyramidal neurons were imaged in consecutive sessions (once per week). The imaging session did not last more than 60 min. The unique pattern of blood vessels was used to re-localize the imaged regions in subsequent imaging sessions. GFP was excited by a femtosecond laser (Spectra Physics) at the wavelength of 880 nm. The intensity of laser and settings of data acquisition were kept constant during experiments. To ensure the dendrites were chosen in amyloid plaque-free regions, methoxy-X04 (1 mg/kg) was intraperitoneally injected 24 h before imaging in the first and last time points. Overview images were taken as 424 × 424 × 350 µm3 (0.83 µm/pixel). From the central regions of these images, apical dendrites (10–80 µm in depth below the surface of brain) were chosen for analysis to make sure the distance between them and amyloid plaques was more than 100 µm. Higher resolution images (0.138 µm/pixel) were used for counting dendritic spines. For illustration purpose, maximal projection images were deconvolved (AutoQuantX3), with contrast and brightness adjusted.
Dendritic spines were analyzed manually in ZEN 2011 (Zeiss) by scrolling through the images in z-stacks. As the limitations of resolution in Z-direction, only laterally protruding spines were counted, as only those could be identified with certainty. In consecutive sessions, a dendritic spine was determined as the same if its location did not change within a range of 0.5 µm along the dendrite. Otherwise, spines that disappeared or emerged compared to the previous imaging session were defined as formed or eliminated, respectively. Spine formation and elimination were normalized into 100 % based on the calculations from the first and second imaging points. The fate of preexisting spines was calculated as the fraction of dendritic spines in the first imaging session that remained stable during the imaging period. Similarly, the fate of new-gained spines was the fraction of formed spines in the first week of EE or matching week of SC that remained stable during the rest of imaging period, indicating how many gained spines incorporated into neural circuits. Transient spines were determined as spines that did not survive more than 1 week and referred to the stability of gained spines in a short-term period.
Following transcardial perfusion with phosphate-buffered saline (PBS) and 4 % paraformaldehyde (PFA), mouse brains were cut into 65 µm thick sections from the somatosensory cortex after being fixed in 4 % PFA overnight. Antibodies against GFAP (Abcam 1:500), Iba1 (Wako 1:500) and beta-amyloid (4G8, BioLegend, 1:500) were used to detect activated astrocytes, microglial, and amyloid beta, respectively. Anti-rabbit Alexa 647 antibody (Invitrogen 1:1000) was used as the secondary antibody. To stain amyloid plaques, sections were incubated with 145 µm methoxy-X04 in PBS for 30 min and then washed with PBS. After mounting on glass coverslips by fluorescence mounting medium (Dako), sections were imaged using LSM 780 confocal microscope (Zeiss). Glial activation, plaque load, or amyloid pathology was quantified as the area with positive staining relative to the cortex area after maximal projection of confocal stacks.
10 % cortical tissues (w/v) were homogenized on ice in lysis buffer with protease inhibitors (Roche), followed by centrifugation at 500 rpm for 1 min. The supernatant was collected and protein concentrations were adjusted by the bicinchoninic acid assay to ensure the same amount of protein being loaded for each sample (80 µg). Samples were mixed with SDS-containing sample buffers and incubated at 100 °C for 20 min. After electrophoresed on 12 % sample gel, proteins were transferred into polyvinylidene difluoride membrane (Millipore). The primary antibodies against IL-1β (Cell signaling), IL-1 RA (Thermo Scientific) and tubulin (Santa Cruz) were used at 1: 1000 concentrations for immunoblotting. Protein bands were quantified by ImageJ.
Quantitative real-time PCR
For gene expression analysis in brain tissue, RNA extraction was performed using TRIzol (Invitrogen). Random hexamer primers, oligo dTs and Superscript II reverse transcriptase (Invitrogen) was used for generation of cDNA. For quantitative real-time RT-PCR, the LightCycler480 system (Roche) and the corresponding SYBR Green detection format was used. Beta-2-microglobulin (β2M) was used as a housekeeper. All analyses were conducted as triplicates. Primers for β2M were designed using Primer3 software. Sequences for the primers were as follows: β2M forward, 5′-TGTCTTTCAGCAAGGACTGG-3′; β2M reverse, 5′-GATGCTGCTTACATGTATCG-3′; IL-1β forward 5′-GGCTGGACTGTTTCTAATGC-3′; IL-1β reverse 5′-ATGGTTTCTTGTGACCCTGA-3′ .
For statistical analysis and comparison, GraphPad Prism 5 was used. In the longitudinal measurements of spine analysis, extra sum-of-squares F test was used when data were fitted with a line using the nonlinear regression. Comparison among groups was performed using one-way ANOVA followed by Newman–Keuls post-test. Two-tailed Student t test was used in comparison between two different groups. The numbers of mice were 4–6 per group for in vivo imaging. 8–12 dendrites were imaged in each mouse. The length of each dendrite was 25–35 µm and the number of spines was normalized to the dendritic length. Data are presented as mean ± SEM. N refers to the number of mice; p < 0.05 was defined as statistically significant (* p < 0.05, ** p < 0.01).
Adaptive structural plasticity of dendritic spines is impaired in deltaE9 mice at the age of 4–5 months
To find out how preexisting neural networks react to the stimulation of EE, we tracked the fate of dendritic spines that existed in the first imaging session over the whole period of enrichment. Interestingly, in control and deltaE9 genotypes, less preexisting spines survived when mice were housed under EE (Fig. 1f, g). This indicated a breakdown of the established neural networks in both groups during EE. Furthermore, the fate of spines that were newly formed in EE or SC was also monitored. A higher number of gained spines remained stable during EE in control mice, but not in deltaE9 mice (Fig. 1h–j). Also, a direct comparison between control and deltaE9 mice revealed that the elimination rate of newly gained dendritic spines induced by EE was higher in the AD mouse model (Suppl. Fig. 2). These results suggest the failure of building up novel neural networks induced by EE in deltaE9 group. Collectively, our data imply that the reorganization of neural networks upon EE is impaired in preclinical stages of AD.
Reduction of BACE1 in deltaE9 mice restores the response with an increase in spine density upon EE
Pioglitazone rescues the deficits of adaptive dendritic spine plasticity in deltaE9 mice
IL-1 RA rehabilitates the impaired adaptive plasticity of dendritic spines in deltaE9 mice
As excitatory postsynaptic compartments, dendritic spines receive and integrate informational input from presynaptic terminals . This function is supposed to be disturbed at the very early stages of AD pathogenesis, which may explain why synaptic loss is a much better indicator for cognitive impairment in AD than Aβ burden or neuronal loss . With the advent of cognitive decline, irreversible damage may have already occurred. Prevention strategies in the asymptomatic stages of AD are therefore warranted.
Preclinical AD is replicated in young deltaE9 mice that develop amyloid deposits before the onset of cognitive decline [4, 14, 27, 34, 50]. In this study, we found that 4–5-month-old deltaE9 mice did not increase dendritic spine density when housed under EE in contrast to control mice. The novel external environment also failed to remodel neural networks in these transgenic mice. Reduction of BACE1 activity in deltaE9 mice reduced the deposition of Aβ and restored the increase of spine density during EE, but not the impaired reorganization of neural networks. However, anti-inflammatory treatments, pioglitazone and IL-1 RA, successfully rescued the spine density increase and neural network remodeling upon EE in deltaE9 mice. These results suggest that neuroinflammation contributes to impaired adaptive plasticity of dendritic spines in preclinical stages of AD.
Structural plasticity of dendritic spines refers to the change of their distribution in response to experience . Learning and sensory experience have been reported to remodel neural connections through de novo growth and loss of dendritic spines, which provides a structural substrate for adaptive behaviors. Spine density increases after spatial learning tasks or manipulations that intensify sensory inputs [26, 28], while deprivation of sensory experience leads to a decrease in spine density . This structural synaptic plasticity may substantially boost information storage capacity in the brain . The failure to increase spine density in young adult deltaE9 mice upon EE suggests an impairment of experience-dependent structural plasticity in asymptomatic stages of AD, before spine loss occurs. In addition, stabilized newly formed spines and destabilized preexisting spines in novel experience reflect a rewiring of neural networks, which facilitates a quicker adaption of brain to the same situation in the future [19, 54, 56]. Interestingly, the ability to dismantle the preexisting neural networks in novel external environment remains intact in deltaE9 mice. However, deltaE9 mice fall short of the establishment of novel neural networks. These results imply that experience-dependent demolition and construction of neural networks are two processes that are independent from each other.
BACE1 initiates the proteolytic process of APP into Aβ, which accumulates to form amyloid plaques. As Aβ is believed to play a central role in AD, BACE1 becomes an attractive drug target. Indeed, partial reduction of BACE1 activity leads to dramatic reductions on amyloid plaque burden and synaptic deficits with a small decrease of Aβ levels in young AD transgenic mice . However, pharmacological inhibition of BACE1 impairs structural and functional synaptic plasticity implying its physiological role in dendritic spines . The boosted transient spines, which contribute to increased spine formation, in deltaE9/Bace +/− mice during EE indicate the maintenance of experience-dependent synaptic rearrangement requires physiological level of BACE1 activity. It still remains unclear, whether BACE1 itself or its substrates are involved in synaptic physiology.
Amyloid deposition, driving neuroinflammation, is associated with activated glial cells and the release of pro-inflammatory cytokines. These soluble mediators, IL-1β in particular, directly and extensively disturb synaptic transmission and plasticity. IL-1β regulates the expression and phosphorylation of glutamate receptors on dendritic spines . The altered sensitivity of receptors to synaptic glutamate modulates synaptic plasticity. In addition, IL-1β disrupts BDNF signaling cascades and thereby prevents activity-driven formation of filamentous actin in spines, which is required for spine structural plasticity . The restorative effects of pioglitazone and IL-1 RA demonstrated herein implicate a deleterious role of IL-1β in experience-dependent spine structural plasticity preceding cognitive impairment in AD.
Of note, numerous clinical studies have demonstrated that anti-inflammatory treatment reduces dementia risk or delay the onset of AD [3, 8, 18], although anti-inflammatory drugs in clinically manifested AD failed to be proven effective [15, 24]. These trials suggest prevention of inflammatory processes is beneficial at the preclinical stages of AD. Our data confirm that neuroinflammation caused impairments of spine structural plasticity is curable by anti-inflammatory treatment in a preclinical mouse model of AD. This finding implies the normalization of adaptive structural plasticity of dendritic spines may correlate with the beneficial effects of anti-inflammatory treatment in preclinical AD patients.
We conclude that our in vivo dendritic spine analysis reveals that neuroinflammation, caused by amyloid deposition, undermines the adaptive changes of neural networks upon novel external environment before the occurrence of dementia, providing new insights for a possible benefit of anti-inflammatory treatments in preclinical AD.
We would like to thank Sonja Steinbach, Eric Griessinger, and Katharina Bayer for their excellent technical support and animal care. Furthermore, we thank Severin Filser for helpful advice in planning the experiments and preparing the manuscript. This work was funded by the German Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung, project 13N12778 and 0316033C and the European commission within the 7th framework (Extrabrain –606950).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interests.
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