Disruption of oligodendrocyte progenitor cells is an early sign of pathology in the triple transgenic mouse model of Alzheimer's disease

1Alzheimer's disease (AD) is a neurodegenerative disease characterised by age-related decline in learning, memory and cognition. Neuropathological hallmarks of AD are amyloid β (Aβ) plaques and neurofibrillary tangles (NFTs). In addition, myelin disruption is prominent in AD, while myelin loss may predict AD onset and neurodegenerative changes in humans, as well as in animal models of AD. Thus, disruption of myelination is a feature of AD, although the underlying causes are unresolved.

2In the CNS, myelin is produced by oligodendrocytes, which are generated from oligodendrocyte progenitor cells (OPCs), also known as NG2-glia or synantocytes. OPCs are identified by their expression of the membrane proteoglycan NG2 (Cspg4), and are the main proliferative cells in the brain. In the adult brain, OPCs slowly divide asymmetrically to form daughter cells that are responsible for OPC self-renewal and for generating new oligodendrocytes for myelin repair and for myelination of new connections important in learning. In addition, OPCs readily respond to CNS pathology by a characteristic morphological remodeling and increased NG2 expression. Notably, OPCs display increased senescence and disruption in the aging brain, and this may be aggravated in human and mouse AD. These studies suggest that age-related changes in OPCs are related to a reduction in myelination in AD.

3The triple transgenic mouse model of AD (3xTg-AD) harbors presenilin 1 (PS1; M146 V), amyloid precursor protein (APP; Swe), and tau (P301 L) transgenes, and displays progressive AD-like pathology and cognitive impairments in an age-related manner. Immunohistochemical, electron microscope and imaging studies have shown myelin disruption in the hippocampus of 3×Tg-AD mice in 6 month-old mice, concomitant with the appearance of Aβ plaques. Here, we have performed a detailed examination of age-related changes in OPC in the hippocampus of 3xTg-AD mouse. Morphological analysis of total cell surface area, total cell volume, somata volume and branching of main processes demonstrated that OPC display a marked morphological atrophy at 6 months in 3xTg-AD. Morphological atrophy was collateral to a decline in the numerical density of OPC daughter cells and decreased myelin immunostaining in the hippocampus. In contrast, at 24 months in 3xTg-AD OPCs undergo a marked morphological hypertrophy and cluster around Aβ plaques with astrocytes. This study identifies complex changes in OPC morphology that are related to the pathogenesis of AD.

4All animal procedures were carried out in accordance with the United Kingdom Animals (Scientific Procedures) Act of 1986 under licence from the Home Office. All efforts were made to reduce the number of animals by following the 3 Rs. The procedure for generating 3xTg-AD mice has been described previously. The 3xTg-AD mouse line harbors the APP Swedish mutations K670 N/M671 L, the presenilin-1 M146 V mutation and the tau P301 L mutation in a C57BL6 mice background, and all 3xTg-AD and non-Tg control mice were obtained by crossing homozygous breeders. The animals were housed in same-sex cages, kept in 12 hours light-dark cycles with free access to food and water independent of the diet. at these ages that the amyloid and tau pathologies emerge resembling the human Alzheimer's disease progression. Mice aged 6 and 24 months were used, because 3xTg-AD mice have been shown to exhibit myelin disruption at 6 months prior to deposition of extracellular Aβ plaques, which are prominent at 24 months of age. Male 3xTgAD and non-transgenic control mice were anesthetized with intra-peritoneal injection of sodium pentobarbital (50 mg kg) at 6 and 24 months of age. The mice were perfused through the aortic arch with 3.75% acrolein (TAAB, Berkshire, UK) in a solution of 2% paraformaldehyde (Sigma, Cambridge, UK) and 0.1 M phosphate buffer (PB) pH 7.4, followed by 2% paraformaldehyde.

5Brains were removed and cut into 4- to 5-mm coronal slabs of tissue containing the entire rostrocaudal extent of the hippocampus. Brain sections were postfixed in 2% paraformaldehyde for 24 hours and kept in 0.1 M PB, pH 7.4. Coronal sections of the brain were cut into 40 μm thickness using a vibrating microtome (VT1000S; Leica, Milton Keynes, UK). Free floating brain sections in 0.1 M PB, pH 7.4, were collected and stored in cryoprotectant solution containing 25% sucrose and 3.5% glycerol in 0.05 M PB at pH 7.4. Coronal sections at levels −1.58 ⁄ −2.46 mm (hippocampus) posterior to the Bregma were selected for immunohistochemistry according to the mouse brain atlas. The sections were incubated for 30 minutes in 30% methanol in 0.1 M PB and 3% hydrogen peroxide (H2O2) (Sigma). Sections were then rinsed with 0.1 M PB for 5 minutes and placed in 1% sodium borohydride (Sigma) for 30 minutes. The sections were then washed with PB profusely before rinsing in 0.1 M Trizma base saline (TS) for 10 minutes. Brain sections were then incubated in 0.5% bovine serum albumin (BSA) (Sigma) in 0.1 M TS and 0.25% Triton (Sigma) for 30 minutes. Sections were incubated for 24 hours at room temperature in primary antibody: rabbit anti-NG2, 1:400 (Millipore); rat anti-MBP, 1:400 (Millipore); mouse anti-Aβ, 1:1000 (Covance). Tissues were then washed 3 times again in TS and incubated with the appropriate secondary antibody (AlexaFluor 488, AlexaFluor 568, 1:400, Life Technologies) diluted in blocking solution for 1 hour at room temperature on an orbital shaker and protected from the light. Following secondary antibody incubation, tissues were washed 2 times with TS and 3 times with PB before being covered with mounting medium and glass coverslips ready for imaging.

6Images were captured using a Zeiss Axiovert LSM 710 VIS40S confocal microscope and maintaining the acquisition parameters constant to allow comparison between samples within the same experiment. Acquisition of images was done with x20 objective for cell counts and analysis of MBP immunostaining; x40 objective was used to examine relationships between OPCs and Aβ plaques; x100 objective was used for OPC 3D reconstruction and morphological analysis, with z-stacks formed by 80–100 single plains with an interval of 0.3 μm.

7Cell counts for OPCs were performed in a constant field of view (FOV) of 100 μm x100 μm in projected images of z-stacks of 10 optic sections with 1 μm interval. Morphological analyses of NG2-glia was performed as previously used for astrocytes in the 3xTg-AD mouse, using the Cell Analyst program and morphological calculations detailed in. In brief, the Cell Analyst program builds a detailed 3-D reconstruction of the cell based on a series of high resolution confocal z-stacks 0.3 μm apart, which defines the precision of the analysis; we used 5 digital filters (average 3 × 3, convolution, gauss 5 × 5, despeckle, simple objects removal) and a threshold of 50 to determine the surface and volume of the NG2-positive OPCs. Relative density of MBP immunostaining was measured from a constant field of view using ImageJ; a threshold was set from negative controls and parameters were kept constant to avoid experimental errors and/or bias. Results are expressed as mean ± SEM and statistical differences were determined by one-way ANOVA and Newman–Keuls multiple comparison post-hoc analysis or unpaired t-tests, as appropriate, using Graphpad Prism5.0.

8Disruption of oligodendrocytes and myelin is an early event in the hippocampus of 3xTg-AD mice, but it is unclear how this is related to age-related changes in OPCs, which are responsible for life-long generation of myelinating oligodendrocytes. To examine this, we performed immunostaining for NG2 and MBP in 6 and 24 month old 3xTg-AD, compared to age-matched controls (Fig. 1). NG2-positive OPCs were widely distributed throughout the hippocampus of both 3xTg-AD and non-Tg mice (Fig. 1Ai-iv), and cell counts indicated there was a decrease in the overall numerical density of OPCs between 6 months and 24 months in both 3xTg-Ad and non-Tg controls (Fig. 1Av, unpaired t-tests). In addition, we observed an age-related decrease in MBP immunostaining between 6 months and 24 months in non-Tg controls (Fig 1Bi, iii), however this decrease occurred earlier in 3xTg-AD at 6 months (Fig. 1Biii), without further change at 24 months (Fig. 1Biv). Quantitative analysis confirmed that MBP immunofluorescence intensity was significantly less in 6-month 3xTg-AD hippocampus compared to age-matched non-Tg controls (Fig. 1Bv; p < 0.001) and equivalent to the level seen in 24-month controls. The results demonstrate a decline in OPCs and MBP with physiological aging, but although OPC numerical density was unaltered in 3xTg-AD, the loss of MBP was significantly accelerated.

9Similarly to stem cells, OPCs self-maintain by undergoing asymmetric division and recently divided cells occur as closely associated duplets or triplets of ‘daughter cells’ that either self-sustain OPCs or proceed to differentiate into oligodendrocytes. Hence, the frequency of OPC duplets/triplets is a measure of cell division and OPC regenerative potential. To examine this in aging, NG2 immunostaining was performed together with nuclear labeling with Hoechst blue to highlight groups of 2 or more juxtaposed OPC daughter cells (Fig. 2). OPC doublets and triplets were prominent throughout the hippocampus in 6 months non-Tg controls, exemplified in the CA1 (Fig. 2A). Quantitation shows a significant decrease in OPC daughter cells between 6 and 24 months in non-Tg controls in the hippocampus overall (Fig. 2B; p < 0.001, unpaired t-tests), and regional analyses demonstrated equivalent decline in the dentate gyrus (DG), and CA1 and CA3 regions (Fig. 2C–E). At 6 months, the number of OPC daughter cells was significantly less in 3xTg-AD hippocampus than age-matched controls (Fig. 2B; p < 0.001), and declined to the level seen at 24-months in controls (Fig. 2B–E). There was no further change in OPC daughter cells at 24 months in 3xTg-AD, which was equivalent to age-matched controls (Fig. 2B–E). The results support a decline in OPC replication in physiological aging that is observed at a younger age in 3xTg-AD.

10Changes in the cellular morphology of OPCs is a characteristic of the response to CNS pathology and has been reported in human AD and mouse models of the disease. To examine whether this occurs in 3xTg-AD, we performed a detailed morphological analysis of NG2 immunostained OPCs in the hippocampus at 6 and 24 months (Fig. 3), using the Cell Analyst program and morphological parameters detailed by, as described previously for astrocytes in the 3xTg-AD model. The results show no significant difference in the morphology of OPCs at 6 and 24 months in non-Tg controls (Fig. 3A and C), also confirmed by quantification of multiple cellular morphological parameters (Fig. 3E–H). In contrast, at 6 months OPCs displayed marked morphological atrophy in 3xTg-AD, compared to controls (Fig. 3A and B), exhibiting a significant decrease in overall cell size and size of cell somata (Fig. 3E–H). Conversely, at 24 months OPCs displayed marked hypertrophy in 3xTg-AD, compared to age-matched controls (Fig. 3C and D), and 6 months 3xTg-AD (Fig. 3B), with significantly larger cell volume and surface area (Fig. 3E and H) and of cell somata volume and surface area (Fig. 3F and G). It is notable that OPC have circular domains in the x-y plane, but are extremely flat in the z-plane, and their somata are not spherical, but instead appear as a ‘fjord-like’ in the x-y planes, and extremely thin in the z-plane (Fig. 2Aii, Aiii), hence surface-volume relationships are not simple cubic as for a sphere. Overall, the surface-volume ratio is ≥10 in OPC, as it is in astrocytes, although OPC are much smaller and the overall values are correspondingly 2–3 times smaller than equivalent measurements in astrocytes. The results demonstrate OPCs go through complex morphological changes in 3xTg-AD that are not observed in natural aging, characterised by shrinkage in early AD, followed by a clear hypertrophy at late stages, indicative of a reactive metamorphosis.

11Neuropathological changes and Aβ plaque load become significantly greater with age in 3xTg-AD mice in AD-relevant brain regions, including the hippocampus. There is evidence from in vitro studies that Aβ is toxic for OPCs and induces morphological condensation. However, we did not observe a decrease in OPC in 3xTg-AD and cells were hypertrophic at 24 months. We therefore performed double immunfluorescence labeling to examine the relations between OPCs and Aβ plaques in 24 months 3xTg-AD (Fig. 4). The results demonstrate that hypertrophic OPCs are adjacent to Aβ plaques throughout the hippocampus (Fig. 4A). Higher magnification imaging in the CA1 hippocampal area demonstrates the intimate relationships between OPCs and Aβ plaques (Fig. 4B–D). Large Aβ plaques are circumscribed by OPCs and infiltrated by their processes (Fig. 4B). OPCs were observed to be located both between multiple plaques that they contact (Fig. 4C), as well as being embedded within Aβ plaques (Fig. 4D). In the cortex, intraneuronal and vascular Aβ immunoreactivity was prominent in 24 months 3xTg-AD (Fig. 5A and C), and OPCs were directly apposed to these, surrounding them with their processes (Fig. 5B and D). The results demonstrate that OPCs are intimately associated with Aβ plaques in 3xTg-AD, consistent with a recent study in human AD.

12In 3xTg-AD, astrocyte hypertrophy is associated with Aβ plaques, and there is evidence astrocytes play a role in the clearance and removal of Aβ. The close associations of OPC with Aβ plaques demonstrated above stimulated us to examine their interrelationships with astrocytes in the 24-month 3xTg-AD hippocampus (Fig. 6). Triple immunostaining for NG2, GFAP and Aβ shows that both astrocytes and OPCs are densely distributed throughout the hippocampus in association with Aβ deposits (Fig. 6A), and the processes of OPCs and astrocytes overlap extensively, contacting and infiltrating proximal Aβ plaques (Fig. 6B). Furthermore, in the hippocampus of 24 months 3xTg-AD we rarely observed cells co-immunostained for NG2 and GFAP (Fig. 6C and D). NG2 positive astrocytes have been reported in human AD in individuals with high Aβ plaque load, and comparison with NG2+/GFAP− OPCs and NG2−/GFAP+ astrocytes in the same field of view in our study indicates NG2+/GFAP+ cells had a morphology more akin to astrocytes than OPCs (Fig. 6D). Our results demonstrate that OPCs and astrocytes cluster around the same Aβ plaques in 3xTg-AD.

13Age-related loss of myelin is a feature of normal aging and has been shown to be exacerbated in human AD and in multiple animal models of AD-like pathology. Here, we confirm previous observations, by showing that compromised myelination is evident at 6 months of age in 3xTg-AD, to a degree comparable to that observed at 24 months in natural aging. The maintenance of myelination in the adult brain is an ultimate function of OPCs, which divide and regenerate oligodendrocytes more slowly in the aging brain. Our data shows an age-related decrease in the number of OPC daughter cells occurred significantly earlier, at 6 months, in 3xTg-AD, suggesting OPC self-renewal is compromised at early stages of AD-like pathology. Notably, OPCs displayed marked cellular shrinkage at 6 months in 3xTg-AD. In contrast, OPCs were hypertrophic at 24 months in 3xTg-AD and, together with astrocytes, were closely associated with Aβ plaques. Our findings indicate complex changes in OPCs are important markers for early and late stages of AD-like pathology, which may be a major factor in myelin loss.

14Imaging studies in 3xTg-AD show pronounced myelin changes in the fimbria, which acts as the major output tract of the hippocampus, and our results support evidence that myelination is reduced in 3xTg-AD. These results in 3xTg-AD mice correlate well with imaging and postmortem studies in human AD demonstrating widespread abnormalities in oligodendrocytes and myelin, including reduced MBP. Indeed, white matter changes are an early feature of human AD, and the premature loss of MBP immunostaining observed in our study is consistent with myelin loss being an early event in AD pathology and cognitive decline. Certainly, myelin disruption is coincident with the earliest signs of cognitive impairment and neuropathology in 3xTg-AD. Furthermore, myelin disruption has been shown to result in axonal and neuronal degeneration, and myelin injury and loss of oligodendrocytes in AD is associated with axon degeneration and amyloid plaques. The causes of myelin loss in AD are likely to include direct Aβ toxicity, as well as glutamate, metabolic and iron dyshomeostasis. Our results indicate disruption of OPCs is another key event that may contribute to myelin loss in early AD.

15At early stages of disease progression in 3xTg-AD, OPCs displayed a striking morphological atrophy, manifested by decreased cell surface area and volume, concomitant with a >30% decrease in OPC daughter cells, a measure of their recent cell division. The asymmetric division of OPCs is critical for self-replication and the life-long generation of new myelinating oligodendrocytes. Hence, the early atrophy of OPCs and apparent decrease in self-renewal indicates disruption of OPCs may be a key factor in the accelerated loss of myelin in 3xTg-AD. Notably, OPCs extend processes to contact glutamatergic synapses in the hippocampus, and glutamatergic signaling promotes OPC proliferation and differentiation. It is significant, therefore, that synaptic dysfunction, in particular disruption of glutamatergic signaling, is an early event in 3xTg-AD, and is a major component of human AD. Astroglial glutamate homeostasis is also dysregulated in 3xTg-AD, which would further exacerbate glutamate signaling onto OPCs. The atrophy of OPC processes is contemporaneous with disruption of synaptic signaling, suggesting this is an important factor in the observed decline in OPC self-renewal and reduced capacity for replacing myelin lost in AD. Overall, OPC numbers did not decline until later ages in 3xTg-AD, implying the accelerated decline in myelination in 3xTg-AD was not directly related to a loss of OPCs, but may reflect reduced differentiation and OPC senescence. At later stages of disease progression OPCs displayed a marked hypertrophy in 3xTg-AD, with increased cell volume and surface area, thicker processes and enlarged cell bodies, characteristic of an injury response in OPCs. Hypertrophic OPCs contacted Aβ plaques together with astrocytes, which have been described previously. These findings are consistent with OPCs being relatively resistant to Aβ, and there is evidence OPCs are involved in Aβ clearance.

16In conclusion, the results demonstrate that OPCs undergo complex changes that are related to accelerated myelin loss in 3xTg-AD. This study highlights the importance of OPCs in AD pathogenesis.

17AB and AR are shareholders in the company GliaGenesis. The authors declare no other conflicts.

18Ilaria Vanzulli: Formal analysis, Investigation, Methodology, Writing - original draft. Maria Papanikolaou: Formal analysis, Investigation, Methodology, Validation. Irene Chacon De-La-Rocha: Investigation, Methodology, Validation. Francesca Pieropan: Investigation, Methodology, Validation. Andrea D. Rivera: Investigation. Diego Gomez-Nicola: Writing - review & editing. Alexei Verkhratsky: Conceptualization, Writing - review & editing. José Julio Rodríguez: Conceptualization, Resources, Software, Writing - review & editing. Arthur M. Butt: Conceptualization, Data curation, Formal analysis, Funding acquisition, Project administration, Resources, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing.