Environmental Enrichment Rescues Functional Deficit and Alters Neuroinflammation in a Transgenic Model of Tauopathy

Abstract

Alzheimer’s disease (AD) is the most frequent neurodegenerative disorder, affecting over 44 million people worldwide. There are no effective pharmaco-therapeutic options for prevention and treatment of AD. Non-pharmacological approaches may help patients suffering from AD to significantly ameliorate disease progression. In this study, we exposed a transgenic rat model (tg) of human tauopathy to enriched environment for 3 months. Behavioral testing at 6 months of age revealed improvement in functional deficits of tg rats reared under enriched conditions, while sedentary tg rats remained severely impaired. Interestingly, enriched environment did not reduce tau pathology. Analysis of neurotrophic factors revealed an increase of nerve growth factor (NGF) levels in the hippocampus of both enriched groups (tg and non-tg rats), reflecting a known effect of enriched environment on the hippocampal formation. On the contrary, NGF levels decreased markedly in the brainstem of enriched groups. The non-pharmacological treatment also reduced levels of tissue inhibitor of metalloproteinase 1 in the brainstem of transgenic rats. Expression analysis of inflammatory pathways revealed upregulation of microglial markers, such as MHC class II and Cd74, whereas levels of pro-inflammatory cytokines remained unaffected by enriched environment. Our results demonstrate that exposure to enriched environment can rescue functional impairment in tau transgenic rats without reducing tau pathology. We speculate that non-pharmacological treatment modulates the immune response to pathological tau protein inclusions, and thus reduces the damage caused by neuroinflammation.

Keywords

Enriched environment nerve growth factor neuroinflammation non-pharmacological intervention tauopathy

INTRODUCTION

Non-pharmacological interventions (physical exercise, cognitive stimulation, and social integration) have gained increasing attention in neuroscience due to their potential to reduce risk or ameliorate progression of various neurodegenerative conditions, such as Alzheimer’s (AD), Parkinson’s, and Huntington’s diseases, as well as multiple sclerosis, migraine, or stroke [1, 2].

Several clinical studies have shown that a healthy lifestyle could improve or maintain cognitive functioning in the elderly population [3 –5]. For instance, a recent 2-year Finnish Geriatric Intervention Study to prevent Cognitive Impairment and Disability multi-domain lifestyle intervention trial indicated positive effects on cognition [6]. Another comprehensive study—Multidomain Alzheimer Preventive Trial, which examined association of physical activity with cognitive function in older people with memory difficulties, indicated amelioration of cognition after regular leisure-time physical exercise [7].

Such a neuroprotective effect of environmental stimulation was experimentally demonstrated and deeply investigated in multiple rodent models mimicking neurodegenerative disorders, and also in experimental animals displaying various forms of traumatic brain injury or stroke [8]. Transgenic rodents bearing major hallmarks of human neurodegenerative disorders also displayed significant benefits after exposure to enriched environment (e.g., objects for climbing, running wheels, toys) [9 –11].

Molecular, biochemical, and morphological analyses of the brains of rodents reared under enriched housing conditions identified various changes including enhanced neurogenesis, gliogenesis, and angiogenesis, improved synaptic plasticity, increased trophic factor expression, improved dendritic arborization, and consequently increased cell survival [12, 13]. Unravelling the mechanisms of how environmental stimulation helps the brain to cope the burden of pathology can broaden therapeutic possibilities in neurodegenerative diseases.

In AD, the exposure to enriched environment (EE) similarly bestows therapeutic benefits, as demonstrated in transgenic mice expressing mutated amyloid-β (Aβ) protein or presenilins [14, 15]. Caution, however, should be exercised in choosing an animal model and interpreting findings. For example, APPswe/PS1dE9 transgenic mice exposed to EE showed dramatic reduction in Aβ deposition in the cortex and hippocampus, and an increased level of Aβ degrading protease [15]. In closely related APPswe and APPswe/PS1dE9 strains, however, a beneficial effect of EE on cognitive functions has been accompanied by an increased level of amyloid plaques in animals reared under EE conditions [14]. These contradictory results can be attributed to different gender of experimental animals used, supported by the assumption that gender strongly influences the response to APPswe overproduction. Importantly, the increase in Aβ pathology in EE-raised transgenic mice did not affect their cognitive performance. This outcome is compatible with the theory of cognitive reserve in human patients: enriched mice built up a cognitive reserve and resilience, and thus were able to withstand a greater degree of pathological insult than their standard-housed counterparts, with less cognitive decline [14].

The impact of the other major pathological hallmark of AD—neurofibrillary tangles and tau protein—has not been fully elucidated under enriched environmental rearing conditions, probably for lack of an appropriate animal model [15]. Tau transgenic rats expressing human truncated tau protein [14, 16] display a variety of neurobehavioral symptoms reflecting the progression of the neurodegenerative process [17] and offer a great opportunity to study detailed changes in the tau-related neurodegenerative cascade and neuroinflammation in animals exposed to enriched environmental conditions and other non-pharmacological therapeutic approaches

To study directly the impact of non-pharmacological interventions on the development of functional impairment and neuropathology driven by the tau pathological cascade, here we exposed tau transgenic rats to EE and performed behavioral, biochemical gene expression analyses. Our results suggest that exposure to EE can ameliorate sensorimotor impairment and impact neuroinflammation without changing the underlying tau pathology.

METHODS

Animals

In this study, SHR72 transgenic (tg) male rats were used together with SHR non-transgenic (non-tg) male littermates as controls. The tg line was generated via overexpression of truncated tau protein (4R tau, aa151-391) under the control of the mouse Thy-1 promoter, as described and characterized previously [16]. The transgenic rat model demonstrates tau hyperphosphorylation in the hippocampus, cortices, and brainstem at the age of 3 months. The animal model displays progressive increase in tau hyperphosphorylation and formation of neurofibrillary tangles [16]. Rats were housed in standard laboratory conditions in plastic cages in a temperature and humidity-controlled environment with a 12/12 h light/dark cycle and with food and water available ad libitum. Efforts were made to minimize the number of animals used. All experiments were performed in accordance with the Slovak and European Community Guidelines, and were approved by the Ethics Committee of Institute of Neuroimmunology (Slovak Academy of Sciences, Bratislava) and the State Veterinary and Food Administration of the Slovak Republic.

Enriched environment

Enriched rats were allowed to explore spacious cages (100×100×40 cm), interact with more social partners (housed in equal numbers/group), and enjoy various novel stimuli: shelters, running wheels, climbing frames, ropes, a water reservoir, and various types of material to gnaw on, all available daily for 4 h. The position of objects was alternated, and novel objects were made available every day. As controls, we used sedentary transgenic and non-transgenic rat males housed in standard laboratory cages as described above.

Two sets of tg rats and non-tg littermates aged 3 months were used for experiments. The first set of animals (n = 40) was used for behavioral and western blot and ELISA analyses, and divided into four groups reflecting different experimental conditions: enriched tg (n = 10), enriched non-tg (n = 10), sedentary tg (n = 10), and sedentary non-tg (n = 10). Two animals from the sedentary tg in group I developed signs of paraparesis of hind legs and were unable to perform behavioral test therefore were excluded from the study. The second set of animals (n = 48) was used for quantitative PCR analysis and divided into four groups: enriched tg (n = 11), enriched non-tg (n = 13), sedentary tg (n = 12), and sedentary non-tg (n = 12). Exposure to EE for all enriched animals was stopped when rats reached 6 months of age. Two animals from the enriched tg (group II) died before the end of experiment (∼1.5 week before end of experiment); therefore, n = 11 in the enriched tg group.

Behavioral analysis

When rats reached 6 months of age, neurobehavioral impairment was quantified using the NeuroScale testing scheme developed for evaluation of functional impairment in SHR72 line [18]. The battery of tests included beam walking tests, prehensile traction test, and hind limb escape extension reflex assessment. Beam walking tests were performed in order to estimate sensorimotor coordination and balance. Rats were required to traverse 200 cm long wooden beams placed 75 cm above the floor. Escape latency and number of hind limb slips were measured using three types of beams with different cross-sections, square (3×3 cm, easiest), rectangular (4×2 cm, 2 cm wide traversing segment), and round (diameter 3.5 cm, most challenging). Prehensile traction test was performed to measure skeletal muscle strength. Rats were allowed to grasp with both forelimbs a 3 mm thick rod placed 75 cm above a box with thick layer of sawdust, and latency to walk was measured. Hind limb escape extension reflex was examined as a marker of neurological impairment. Healthy rats display an extension of their hind limbs, whereas tg rats display gradual impairment of the response, culminating in a “crossing” or “clasping” phenotype. Test results were converted into points and added up to yield a NeuroScale score.

Sample collection

Rats were deeply anesthetized and humanely euthanized by cervical dislocation. Brain specimens were immediately sampled, frozen in liquid nitrogen, and stored at –70°C. For the postmortem analysis the following regions were sampled: brainstem area due to abundance of neurofibrillary pathology, cortical tissue due to extensive manifestation of hyperphosphorylated tau, and hippocampus as a region responsible for various cognitive skills and motor behaviors.

Total protein extraction

For total protein extraction, samples were processed as previously described [19]. Briefly, tissues were homogenized in ice-cold extraction buffer supplemented with complete protease inhibitors without EDTA. Homogenates were incubated on ice for 5 min and cleared by centrifugation at 20,000 g for 20 min at 4°C. The supernatant was collected and stored at –20°C until further use. Concentrations of the samples were estimated using Bradford method (Bio-Rad laboratories GmbH, Munchen, Germany) according to manufacturer’s instruction.

Western blot analysis

20 μg of total protein extract was separated using SDS-PAGE and transferred to nitrocellulose membranes, Ponceau staining was used to compare equal loading (Supplementary Figure 1). The membranes were probed with primary antibodies: pan-tau monoclonal antibody DC25 recognizing residues aa347-354 (Axon Neuroscience, Bratislava, Slovakia), TAU1 (de-phosphorylated Ser¹⁹⁵/Ser¹⁹⁸/Ser¹⁹⁹/Ser²⁰²; Sigma-Aldrich, Bratislava, Slovakia), or AT8 (pSer²⁰²/pThr²⁰⁵; Thermo scientific, Rockford, IL, US). After washing, membranes were immunostained using respective secondary antibodies (Dako, Glostrup, Denmark), developed using an enhanced chemiluminescence kit (Thermo Scientific, Rockford, IL, US), and the signal was detected using a LAS3000 imaging system (FUJI Photo Film Co., Ltd, Tokyo, Japan). Recombinant human 6 tau isoforms and recombinant truncated tau aa151-391/4R were used as positive controls for brainstem samples. For hippocampi samples, only recombinant 6 tau isoforms were used as controls. Densitometry analysis of blots was performed using Advanced Image Data Analyzer software (AIDA Biopackage Raytest, Germany). The levels of AT8 and Tau1 were normalized to total tau (DC25) levels.

ELISA measurements of neurotrophic and inflammatory factors

Concentrations of neurotrophic and inflammatory factors in the brainstem and hippocampus were measured using ELISA kits: Quantikine Rat TIMP-1 Immunoassay, Quantikine Mouse CCL2/JE/MCP-1 Immunoassay, Quantikine Mouse IGF-1 Immunoassay (R&D Systems, UK), NGF Emax^® Immunoassay System, GDNF Emax^® Immunoassay System, BDNF Emax^® Immunoassay System, and NT-3 Emax^® Immunoassay System (Promega, US) according to the manufacturer’s instructions. To assay total NGF, acidification and neutralization of the sample was performed.

RNA extraction and quantitative PCR

Total RNA was isolated by TRI Reagent extraction method according to the manufacturer’s protocol (Sigma-Aldrich, US). Resulting RNA was briefly air-dried, dissolved in 100 μL of RNAse-free water (Qiagen, Germany). RNA samples were kept frozen at –80°C. Integrity of isolated total RNA samples was determined by Agilent 2100 Bioanalyzer using a RNA 6000 Nano Labchip kit (Agilent Technologies, US). For quantitative PCR analysis we have used high quality RNA samples (RNA Integrity Number; RIN = 8.5–9.6). Synthesis of the first strand was carried out using the High capacity cDNA reverse transcription kit (Applied Biosystems, US) according to the manufacturer’s recommendations. Briefly, 10 μL of the 2x reverse transcription master mix was mixed with 1 μg of RNA sample and cDNA was synthesized under the following conditions: 25°C for 10 min, 37°C for 120 min, 85°C for 5 min and 4°C for 1 min. Levels of Rt1-Ba, Cd74, Gpnmb, Lgals3, Il-1β, Tnf-α, and Il-6 mRNA were determined using qPCR with Gapdh as a standard. Following TaqMan primer assays (FAM for target genes and VIC for reference gene) were used for gene expression analysis: Rt1-Ba (Rn01428455_g1), Cd74 (Rn00565062_m1), Gpnmb (Rn00591060_m1), Lgals3 (Rn00582910_m1), Il-1β (Rn99999009_m1), Tnf-α (Rn00562055_m1), Il-6 (Rn01410330_m1); Gapdh (Rn99999916_s1) (Applied Biosystems). Composition of the quantitative PCR reaction (25 μL) was as follows: 12.5 μL 2×TaqMan gene expression master mix; 1.25 μL 20× target FAM- or VIC-labelled TaqMan primer assay; 10.25 μL nuclease-free H₂O and 1 μL cDNA sample (50 ng/μL). PCR reactions were performed in duplicate under the following conditions: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Comparative ddCt analysis was performed in order to compare gene expression between sedentary and enriched-treated tg and non-tg animals. Results are expressed as a fold change of target gene mRNA level in animals compared to average of the sedentary non-tg rat group.

Statistical analysis

Statistical analysis was performed using Prism (Version 6, GraphPad Software, Inc., US). Data were analyzed using two-way ANOVA followed by Bonferroni’s post hoc test for multiple group comparison. Bartlett’s test for homogeneity of variances was performed for all analyses followed by multiple comparison (Supplementary Material 1 and 2). For the comparison between individual groups, t-test was used. For immunoblots, an unpaired t test was used. Data sets are expressed as mean±standard error mean (SEM). Differences were considered to be statistically significant if p < 0.05. *p < 0.05, **p < 0.01 and ***p < 0.001 are used to denote statistical significance.

RESULTS

Enriched environment delayed functional impairment in tau transgenic rats

Behavioral testing at 6 months showed that environmentally enriched tg rats outperformed rats kept in standard housing conditions, and were behaviorally indistinguishable from non-tg littermates across the NeuroScale battery (Table 1) (CI are provided in Supplementary Material 2). In the beam walking test, enriched tg animals walked across the beams of different cross-sections faster than sedentary tg rats: squared 3×3 cm beam (p = 0.0002; Fig. 1A), rectangular 4×2 cm beam (p = 0.0004; Fig. 1B), and round 3.5 cm beam (p≤0.0001; Fig. 1C). Interestingly, there was variability in the latency to walk between tg rats in the sedentary group, while in the enriched group this variability completely disappeared. Non-tg animals kept under conditions of EE passed the beams of all sections faster than sedentary non-tg animals: squared 3×3 cm beam (p < 0.0001; Fig. 1A), rectangular 4×2 cm beam (p = 0.001; Fig. 1B), and round 3.5 cm beam (p < 0.0001; Fig. 1C).

Table 1

Results from behavioral analysis

	Transgenic factor			Enrichment			Interaction
Parameters	df	F	p	df	F	p	df	F	p
Beam walking 3×3 cm (latency)	1	17.5	0.0002	1	27.2	<0.0001	1	15.89	0.0004
Beam walking 3×3 cm (slips)	1	29.44	<0.0001	1	34.18	<0.0001	1	26.77	<0.0001
Beam walking 4×2 cm (latency)	1	15.65	0.0004	1	23.20	<0.0001	1	12.66	0.0012
Beam walking 4×2 cm (slips)	1	24.42	<0.0001	1	28.69	<0.0001	1	24.5	<0.0001
Beam walking 3,5 cm (latency)	1	21.19	<0.0001	1	27.83	<0.0001	1	15.42	0.0004
Beam walking 3,5 cm (slips)	1	145.6	<0.0001	1	139.2	<0.0001	1	93.21	<0.0001
Hind limb reflex (score)	1	198.7	<0.0001	1	7.385	0.0105	1	6.560	0.0153
Prehensile traction (latency)	1	10.85	0.0024	1	4.167	0.0495	1	1.180	0.2854
NeuroScale (score)	1	41.48	<0.0001	1	46.36	<0.0001	1	34.42	<0.0001

Two-way ANOVA followed by Bonferroni’s post hoc was used for statistical analysis.

Fig.1

Exposure to enriched environment prevented sensorimotor decline in transgenic rats. Decreased latency to walk across beams of different cross-sections: squared 3×3 cm (A), rectangular 4×2 cm (B), and round 3.5 cm (C); and number of slips: squared 3×3 cm (D), rectangular 4×2 cm (E) and round 3.5 cm (F) in 6-month-old tg rats housed under EE (n = 10) compared to sedentary tg rats (n = 8) housed under standard conditions. Enriched tg rats displayed increased latency to walk off in prehensile traction test (G). Hind limb extension reflex of enriched tg rats was slightly improved compared to sedentary tg littermates (H). Overall NeuroScale score reflected functional impairment of sedentary tg rats and sensorimotor improvement of enriched tg rats (I). In each panel, statistical significance is shown only between sedentary tg and EE tg rats. Statistical analysis was performed using two-way ANOVA followed by Bonferroni post hoc. Data are shown as mean±SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

The number of slips indicating the balance and coordination difficulties was lower in tg animals exposed to EE in comparison with tg rats housed in standard cages: squared 3×3 cm beam (p < 0.0001; Fig. 1D), rectangular 4×2 cm beam (p = 0.0001; Fig. 1E), and round 3.5 cm beam (p < 0.0001; Fig. 1F). However, statistical analysis did not reveal differences between non-tg (control) groups in number of slips during passage across the beams: squared 3×3 cm beam (p = 0.0734; Fig. 1D), rectangular 4×2 cm beam (p > 0.9999; Fig. 1E), and round 3.5 cm beam (p = 0.1029; Fig. 1F).

Prehensile traction test analysis indicated weakness of skeletal muscles of sedentary tg rats compared to enriched transgenes (p = 0.024; Fig. 1G), but not in non-tg animals (p = 0.5678). Compared to non-tg animals, both tg groups displayed impairment in hind limb escape extension reflex (p < 0.0001); however, the enriched tg rats scored slightly better than the sedentary tg (p = 0.0129; Fig. 1H). Overall, sharp decrease in NeuroScale score in transgenic animals exposed to EE compared to sedentary tg animals (p < 0.0001) reflected amelioration of sensorimotor dysfunction. The NeuroScale score of sedentary controls (non-tg) was similar to the enriched non-tg group (p = 0.567, 95% CI = –6.328 to 11.76) (Fig. 1I).

Enriched environment did not affect tau phosphorylation pathway

We assessed the possible influence of EE on tau phosphorylation in transgenic rats. Using western blot analysis, we estimated the levels of tau protein and its phosphorylation status at an AD-relevant epitope (pS202/pT205) in the brainstem and hippocampus of enriched and sedentary tg rats using antibodies AT8 (pS202/pT205), TAU1 (non-phosphorylated at S202/T205), and DC25 (against total tau). We observed a slight reduction in tau phosphorylation detected by the AT8 antibody in the brainstem of enriched tg rats. However, we found no prominent differences in tau phosphorylation between enriched and sedentary tg rats (Fig. 2). Statistical analysis revealed no significant difference in the levels of AT8 and TAU1 between the two groups in the regions analyzed (Hippocampus: AT8 p = 0.342; TAU1 p = 0.698; Brainstem: AT8 p = 0.896; TAU1 p = 0.342, respectively).

Fig.2

Western blot analysis did not reveal an effect of enriched environment on total tau protein levels and phosphorylation at AD-relevant epitope (pS202/pTh205). Representative western blot analysis of phospho-tau levels in brainstem and hippocampus of enriched tg rats (n = 4/group) (1–4, 6-month-old males) and sedentary tg rats (c1–c4, 6-month-old males). In the brainstem samples, recombinant human 6 tau isoforms and truncated tau 151-391/4R were used as a positive control; in the hippocampi samples, only the recombinant 6 tau isoforms (+c) were used as a positive control. DC25 (total tau) was used to normalized the levels of AT8 or Tau 1 antibodies. Statistical analysis using unpaired t test revealed no difference in the levels of AT8 or TAU1 levels between the two groups in both regions examined.

Enriched environment altered levels of NGF in specific regions

To reveal the impact of increased physical, mental, and social stimulation on levels of neurotrophic factors in the brainstem and hippocampus, we measured concentrations of nerve growth factor (NGF), insulin-like growth factor (IGF1), brain-derived neurotrophic factor (BDNF), and glial cell line-derived neurotrophic factor (GDNF).

EE increased the levels of NGF in hippocampus of tg (p < 0.0001), as well as non-tg animals (p < 0.0001). The brainstem of sedentary tg rodents had lower levels of NGF when compared to sedentary non-tg animals (p < 0.0001). However, in the brainstem, both tg (p < 0.0001) and non-tg (p < 0.0001) rats exposed to EE showed significantly lower NGF levels compared to their sedentary counterparts (Fig. 3A). Interestingly, levels of most of the measured trophic factors remained stable after EE in both tg as well as non-tg animals: level of IGF1 in brainstem (tg: p = 0.7933, non-tg: p = 0.5259) and in hippocampus (tg: p = 0.5727, non-tg: p = 0.5421) (Fig. 3B), level of GDNF in brainstem (tg: p = 0.1433, non-tg: p = 0.5888) and in hippocampus (tg: p > 0.9999, non-tg: p > 0.9999) (Fig. 3C), level of BDNF in brainstem (tg: p > 0.9999, non-tg: p = 0.5573) and in hippocampus (tg: p > 0.9999, non-tg: p = 0.8193) (Fig. 3D).

Fig.3

Enriched environment affected levels of neuronal growth factor. Levels of NGF were decreased in the brainstem and increased in hippocampus of both tg and non-tg animals kept under enriched environment (A). Levels of IGF-1 (B), GDNF (C), and BDNF (D) were unaffected by housing method. Data were analyzed using two-way ANOVA followed by Bonferroni post hoc. Data are represented as mean±SEM. ***p < 0.001. (n = 10 for all groups except sedentary tg where n = 8).

Enriched environment altered levels of TIMP1 but not MCP1

We have previously reported that TIMP metallopeptidase inhibitor 1 (TIMP1) and monocyte chemoattractant protein-1 (MCP1), markers of microglial activity, are upregulated in the brainstem, but not in the hippocampus of the transgenic rodent model of tauopathy [20, 21]. Therefore, we analyzed the levels of TIMP1 and MCP1 in the brainstem, in order to see whether the EE had any influence on activation of inflammatory processes accompanying neurodegeneration in the tg rat model. The level of TIMP1 in the brainstem was increased in both enriched and sedentary transgenic rats compared to their non-transgenic counterparts. However, TIMP1 level was lower in enriched than in sedentary tg rats (p < 0.001). In non-tg control rats, we detected no statistically significant effect of EE (p = 0.5259) (Fig. 4A). There was no difference in the levels of MCP1 between sedentary tg and non-tg rodents (Fig. 4A). The level of MCP1 remained unaffected by enrichment in both groups of animals (tg: p = 0.5483, non-tg: p = 0.9316) (Fig. 4B).

Fig.4

Enriched environment affected levels of TIMP-1 proinflammatory factor. Levels of TIMP1 decreased in the brainstem of enriched tg animals compared to sedentary tg rats (A). MCP1 levels remained unchanged in the brainstem (B). Statistical analysis was performed using two-way ANOVA followed by Bonferroni post hoc and shown as mean±SEM. ***p < 0.001. (n = 10 for all groups except sedentary tg where n = 8).

Enriched environment altered the pattern of neuroinflammation

Gene expression analysis of brainstem area that is predominantly affected by neurofibrillary degeneration in tg rats revealed prominent upregulation of inflammatory markers Rt1-Ba (2.5-fold, p = 0.004), Cd74 (2.9-fold, p = 0.0004), Gpnmb (3.3-fold, p < 0.0001), Lgals3 (2.8-fold, p = 0.0002), Il-1β (2.7-fold, p = 0.0034), and Il-6 (1.5-fold, p < 0.0001) in sedentary tg animals when compared to non-tg sedentary controls (Fig. 5A–F). Exposure of animals to EE potentiated this effect in tg rats and resulted in increased expression of MHC class II molecules Rt1-Ba and Cd74 (1.44-fold, p = 0.0466 and: 1.41-fold, p = 0.0302 respectively), and markers of phagocytic macrophage activity Lgals3 (1.32-fold, p = 0.0490) and Gpnmb (1.45-fold, p = 0.0106) (Fig. 5A–D). None of these factors were influenced by enrichment in non-tg animals (Rt1-Ba: 0.95-fold, p > 0.9999, Cd74 : 0.96-fold, p > 0.9999, Lgals3 : 1.3-fold, p > 0.9999, Gpnmb: 0.96-fold, p > 0.9999) (Fig. 5A–D). The levels of Il-1β, Il-6, and Tnf-α were slightly elevated in tg animals regardless of enrichment of the environment, but was not significant (Fig. 5E–G). In non-tg animals, EE also had no effect on Il-1β, Il-6, and Tnf-α (Fig. 5E–G).

Fig.5

Enriched environment altered inflammatory signaling. Analysis of inflammatory genes showed elevated expression of Rt1-Ba, Cd74, Gpnmb, and Lgals3 genes in brains of truncated tau-expressing animals. Enriched environment further increased expression levels of these genes in tg animals, while expression in non-tg control animals remained stable at basal levels (A–D). Exposure to enriched environment did no change the expression of Il-1β, Il-6, and TNF-α (E). Statistical analysis was performed using two-way ANOVA followed by Bonferroni’s multiple comparisons test. Data are shown as mean±SEM. *p < 0.05. (n = enriched tg: 11; enriched non-tg: 13; sedentary tg and non-tg: 12 each/group).

We conclude that exposure to EE moderately increased the expression of MHC class II molecules and markers of phagocytic macrophages in tg animals, but did not affect these markers in non-tg (control) animals. Furthermore, levels of pro-inflammatory molecules remained unaffected by enrichment in both tg and control groups, meaning that in tg animals it remains elevated compared to non-tg controls.

DISCUSSION

AD is the most common cause of dementia [22] with no effective pharmaco-therapeutic options currently available. Non-pharmacological approaches, however, may ameliorate clinical signs of disease progression and help patients suffering from AD [23].

Several clinical studies focusing on the non-pharmacological prevention of dementia have shown that a healthy lifestyle (including exercise, healthy diet, cognitive training, and vascular risk management) could improve or maintain cognitive functioning in the elderly population [24]. For instance, the recent Finnish Geriatric Intervention Study to Prevent Cognitive Impairment and Disability recruited 1,260 participants aged 60–77 years and demonstrated that a multi-domain intervention, including diet and physical activity, postponed/prevented memory difficulties in at-risk elderly participants [6]. Another study, the Multidomain Alzheimer Preventive Trial, showed that leisure-time physical activity improved cognitive functions in older people claiming memory difficulties; however, physical activity in the form of housework did not have an effect on cognition, and gardening showed mixed results [25].

Enriched environment has been used as a tool for physical, mental, and social stimulation of animal models, i.e., as an equivalent of healthy lifestyle in humans, and its impact has been explored in a variety of transgenic rodent models of AD. A positive behavioral effect of EE at various rodent models of neurodegenerative disorders has been demonstrated by numerous studies. For instance, enriched housing had a positive effect on motor coordination and decreased the number of trials needed to reach the landmark water maze criterion in Lurcher mutant mice [26]. Similarly, PS1/PDAPP mice expressing human mutant presenilin-1 and the amyloid precursor protein displayed better performance in multiple cognitive tasks (Y-maze, Morris water maze, circular platform, etc.) after enrichment of the environment [27]. Triple-transgenic AD mice (APP Swedish, MAPT P301L, PSEN1 M146V) at advanced stage of disease progression showed improvement in spatial learning and working memory after EE [28]. On the other hand, no rescue of working memory deficits upon EE has been reported in APP/PS1KI mice [7, 29, 30].

We studied the impact of EE in SHR72 rat line, a severely affected transgenic rat model of tauopathy expressing human truncated protein tau [31]. This line develops the entire cascade of neurodegeneration, including formation of neurofibrillary tangles accompanied by severe neuroinflammation in the brainstem and spinal cord. To obtain a detailed description of the impact of physical, mental, and social stimulation on the progress of neurodegeneration in the tg line, we have performed comprehensive measurements of behavioral and biochemical analysis. Exposure to EE ameliorated sensorimotor impairment of tg rats, which performed most tests comparably to their non-tg controls.

We then performed analysis of total tau levels in the brainstem and hippocampus, and assessed the level of phosphorylation of the AD-relevant epitope pS202/pTh205. The tg rats start to develop neurofibrillary pathology in the brainstem at 5-6 months of age, and show reduced lifespan of 7-8 months [16]. We asked whether exposure to EE before neuropathology worsens could promote the activity of clearance systems in the neuronal environment and thus lead to a decrease in tau levels in the brains of tg rats. Previous studies have demonstrated that EE rescues tau hyperphosphorylation [11 , 33]. Our study also shows decrease in tau phosphorylation, which, however, was insignificant. Since we used animals of the same age and same social cohort throughout the experiment, we eliminate the influence of social stress in our experimental group. We have reported that genetic background influences the manifestation of neurofibrillary pathology and inflammation [34]. Moreover, it is also shown that gender of the animal also varies the response to environmental stimulation [15]. Therefore, factors such as genetic background and gender of the animals may influence of environmental stimulation in our study. However, our results show that EE can improve sensorimotor abilities in tg rodents despite retaining neuropathological characteristics of tauopathy.

Increased expression of various trophic factors following short- or long-term exposure of experimental animals to EE is considered to be a major causative factor of improvements observed at the biochemical, structural, anatomical, and ultimately functional levels. The upregulation of GDNF, BDNF, NGF, neurotrophin-3, IGF-1, and vascular endothelial growth factor is responsible for the development, differentiation, and survival of neurons, while providing communication among nerve and glial cells [35]. On the other hand, it is observed that the EE induced changes in these factors may be due to regional differences [36]. We measured changes in concentrations of several trophic factors in response to regular short-term exposure to EE in order to estimate their possible role in amelioration of truncated tau-induced impairments.

BDNF is a trophic factor expressed predominantly in the hippocampus, and exercise-induced increase in BDNF concentrations has been associated with improvements in cognitive functions. We observed, however, that the levels of BDNF remained unchanged, both in sedentary and enriched transgenic rats. Several reports show increase in levels of BDNF after EE [32, 36], in some case region-dependent [36]. However, behavioral improvements without changes in BDNF levels have been reported earlier [10]. Our result also suggests that EE may not always affect BDNF levels in rodents.

Another important trophic factor, NGF, is essential mainly for survival and differentiation of sensory and sympathetic neurons in the peripheral nervous system [37], whereas in the CNS, NGF distribution correlates with cholinergic nerve distribution [38]. After exposure to EE, NGF levels decreased in the brainstem and increased in the hippocampus of enriched tg rats. The enrichment-induced increase in hippocampal NGF is in accordance with previously published studies [39 –41] and may explain the improved sensorimotor outcome of enriched non-transgenic and transgenic rats, since NGF is primarily responsible for cell stimulation, survival, and cell proliferative functions [39]. In the brainstem, the levels of NGF were reduced in both enriched and sedentary transgenic rats. Furthermore, sedentary tg rats displayed lower NGF levels compared to age-matched sedentary non-tg rats in the brainstem. Interestingly, the role of NGF in the brainstem is least investigated. However, NGF is known to ameliorate tissue inflammation [42, 43]; therefore, we speculate that the reduction in NGF may be one of the factors responsible for the proinflammatory environment in the brainstem of the tg rat model. On the other hand, the exposition of animals to EE conditions unexpectedly lowered the levels of NGF even further also in non-tg rats, erasing the difference between tg and non-tg animals. Interestingly, exposure to EE reduced the levels of NGF in cortical areas when compared to socially isolated rats, but did not affect the levels of NGF in the hippocampus [44]. Therefore, we speculate that the overall reduction in NGF levels in the brainstem of enriched rats may be region-specific, and is independent of the pathology in the tg rodents. Elaborate studies are necessary to delineate the role of NGF in brainstem, and the consequence of EE on NGF in the rodents.

Finally, we analyzed the impact of EE on inflammatory pathways. Our results revealed an upregulation of markers of microglia and macrophages, whereas enrichment did not affect the levels of pro-inflammatory cytokines, which remained elevated in tg animals, in comparison to wild-type controls. We have previously shown that human truncated tau promotes upregulation of immune molecules and morphological activation of microglia in tg rats. In parallel, MHC class II positive blood-borne monocytes are attracted to and pass through the blood-brain-barrier into the regions of the brain that are affected by tau pathology [21]. Importantly, only a limited number of microglia express MHC class II molecules in tg rats, and thus the genetic background of tg rats expressing human truncated tau likely determine the inflammatory pattern induced by the tau neurodegenerative pathway [45]. The elevated expression of Rt1-Ba and Cd74 markers following exposure to EE could be explained by an increased number of microglia, overstimulation of microglia, or increased influx of blood-born monocytes into the brain [21]. Rt1-Ba and Cd74 are two genes involved in the MHC II pathway responsible for antigen processing, and we speculate that this pathway is linked to increased phagocytic activity of microglia, which is in line with upregulation of Lgals3, a marker of phagocytic microglia and a chemo-attractant for monocytes and macrophages [46, 47]. We have previously reported that neurofibrillary degeneration is associated with increased expression of Il-1β and Tnf-α in the brain [20]. Here we demonstrated further that EE had no effect on already elevated levels of Il-1β, Il-6, and Tnf-α in a truncated tau-expressing line.

Pro-inflammatory signaling is also co-regulated by a feedback mechanism that involves overexpression of Gpnmb. GPNMB protein has an important role in sustaining the phagocytic activity of immune cells and acts as a suppressor of innate immunity, inhibiting the activity of macrophages and microglia by negative regulation of pro-inflammatory molecules, such as Tnf-α, Il-6, and Nos2 [48, 49]. The expression of Gpnmb was elevated after exposure to EE, and we hypothesize that EE also potentiates anti-inflammatory signaling that controls the neuroinflammation in tauopathy.

The data sets from behavioral test and the inflammatory markers were associated with larger and non-homogeneous variances in different groups, as stated in the Supplementary Material (Bartlett’s test for homogeneity of variances). Such non-homogenous variances could influence the statistical results as computed by ANOVA, namely somewhat inflating type-1 errors in respective analyses.

We suggest that sensorimotor stimulation provided by EE leads to profound activation of pathways of innate immunity involving enhanced antigen presentation and increased phagocytic activity of immune cells, specifically under neurodegenerative conditions. The close relationship between immune system, brain function, and EE is motivating efforts toward formulating non-pharmacological therapies for treating neurodegenerative disorders. Our study demonstrates that EE can be used as an alternative approach to delay the progression of disease pathogenesis in AD and other neurodegenerative tauopathies.

Footnotes

ACKNOWLEDGMENTS

The authors thank the staff of animal house, Institute of Neuroimmunology for their technical assistance. This work was supported by research grants APVV-18-0515, APVV-17-0642, VEGA 2/0118/19 and VEGA 2/0110/20.

Authors’ disclosures available online ().

The supplementary material is available in the electronic version of this article: .

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