Background
Negative cognitive effects are known to be an adverse effect of anticholinergic (AC) drugs and are assumed to be transient and reversible [
1‐
3]. However, there is a new emerging hypothesis for the connection between AC exposure and pathogenesis of Alzheimer’s disease (AD), with the primary clinical lead for this connection being Parkinson’s disease (PD) [
4,
5]. Trihexyphenidyl (THP) is the most commonly used AC drug in PD patients. Perry et al. found that continuous THP use for at least 2 years doubled the prevalence of both amyloid plaque and neurofibrillary tangle densities in PD patients [
4‐
6]. Furthermore, some animal studies demonstrated that use of ACs increased Aβ peptide presence in the cortex and hippocampus, while selective M1 agonists are efficacious for AD treatment [
7,
8]. Therefore, duration of AC drug administration has been described as a risk factor for appearance of dementia in PD patients. Despite early epidemiological evidence supporting this hypothesis in the clinic, understanding of the impact of ACs on pathogenesis of neurodegeneration is currently limited. In particular, it is not clear whether the onset of events that results in AC administration is part of aging or reflects early AD development. Understanding such processes is vital as most drug therapies involve chronic administration.
In the current study, we examined the hypothesis that duration of AC drug exposure affects progression of central nervous system (CNS) neurodegeneration. We treated rats with THP (0.3 or 1.0 mg/kg/day, intraperitoneal (IP)) for 7 months; these concentrations have proven successful in previous studies [
9,
10]. Administration was started in 3-month-old normal Sprague-Dawley (SD) rats, while the control group was provided with normal saline (NS) only. Behavioral performance was assessed using the Morris water maze (MWM) and open field tests. Additionally, after behavioral testing, whole genome oligo microarrays and quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) were performed in the hippocampus to gain insight into the global gene expression and pathway responses to long-term THP exposure. Further, the cerebral cortex and hippocampus were histologically examined for associated neurodegeneration. We found that THP treatment altered neuroimmune responses and promoted CNS neuroinflammation features consistent with microgliosis and microglia activation. We believe that such detailed information is critical for understanding the long-term effects of ACs on occurrence or earlier development of AD.
Discussion
Experimental and clinical studies consistently show that cholinergic system dysfunction has a detrimental impact on cognitive performance. Clinical studies suggest that AC agents may produce cognitive side effects among older adults, and duration of AC administration has been described as a risk factor for the appearance of dementia in PD patients. The putative mechanisms may be associated with dysregulation of cholinergic neurotransmission, thereby leading to memory impairments. Nevertheless, the exact mechanisms remain unclear. Here, we report novel findings that long-term THP administration disrupts neuroimmune signaling, with transcriptional activity of antigen processing and presentation noticeably downregulated in the hippocampus of rats. Additionally, we found exacerbated neuroinflammation with misfolded tau protein pathology related to neurodegeneration.
Aging is a multifactorial process that leads to altered behavioral function, including learning and memory. Here, we assessed AC treatment duration as a risk factor for cognitive performance, with THP administration for more than 7 months in aging rats. Thus, we assessed behavioral performance of NS-aging and THP-treated, aging rats in the MWM and open field tasks. The MWM is a task that is commonly used to assess rodents’ spatial learning and memory ability, and the normal function of the hippocampus is essential for this task [
17,
18]. In the present study, animals were tested in place navigation and probe trials. It should be noted that only rats treated with THP (1.0 mg/kg, IP) displayed a significantly poor performance in spatial tasks during the initial 3-month training session, in which escape latency was significantly disrupted. It is known that THP acts as a muscarinic receptor antagonist, and muscarinic acetylcholine receptors play key roles in facilitating cognitive processes. This impairment is consistent with several studies using nonselective muscarinic receptor antagonists to block cholinergic signaling in aging and AD rodent models [
19,
20]. However, we did not find memory disruption among THP-dosed and NS age-matched groups upon repetition of MWM training following subsequent treatments, with escape latency or swimming speed showing no significant impairment. Moreover, after the final place navigation training, all tested groups generated similar path length percentages during the probe task, while the THP (0.5 mg/kg) group spent significantly more time in the target quadrant of the apparatus compared with the other two groups. The probe data confirm that memory disruption is only present early on in THP treatment. In addition, after the MWM test, THP- and NS-treated rats showed no significant behavioral discrepancies in the open field test. THP and the behavioral deficit in memory retrieval at the start of place navigation may reflect its acute function in blocking muscarinic receptors.
Using microarray analysis, the global impact of THP on the hippocampal gene expression was examined. GO and KEGG pathway analysis showed that the most significantly affected GO category for long-term THP treatment was
immune response and antigen processing and presentation or
neuroimmune/neuroinflammatory functions. In particular, genes related to the function of the MHC class I protein complex were significantly downregulated (Table
2 and Fig.
4b). These data were verified by qRT-PCR and confocal-immunofluorescence analyses, with MHC-related genes and MHCI expression significantly downregulated in the rat brain (Fig.
5a, b). Hence, our microarray findings show that long-term THP treatment suppresses innate and adaptive immune homeostasis in laboratory animals. This indicates that THP may function either directly or indirectly in several immune responses and neuroinflammation.
Next, we found that subchronic THP exposure could prime pronounced neuroimmune/neuroinflammatory dysfunction and neuronal phenotypic changes. Neuroinflammatory abnormalities represent a key difference between THP- and NS-treated, age-matched rats. Specifically, THP-treated rats display significantly more Iba1- and CD11b-positive cells of phagocytic morphology compared with age-matched NS-treated littermates. Immunoblotting confirmed the increase in CD11b- and CD68-positive cells associated with long-term systemic THP challenge.
Notably, our current study shows that in activated microglia, THP specifically upregulates M1 muscarinic receptor expression, with a concomitant increase in CD68 (Fig.
9c). THP is widely used as a nonselective muscarinic antagonist and inhibits acetylcholine transmission via blockage of mAChRs [
7,
8]. Our current findings suggest that long-term THP use may upregulate muscarinic M1 receptor expression on microglia, and thereby play a role in stimulating phenotypic changes in microglia. Although we cannot exclude the involvement of other muscarinic receptor types in the observed THP effects on microglia, our results support our previous links between cholinergic modulation and the immune system and inflammation.
Involvement of inflammation is considered to be an important factor in resistance variability or susceptibility to AD [
21,
22]. Here, we found different alterations in tau pathology spreading in an AD-like manner. In normal young rat brain (3 months old), there was less tau hyperphosphorylation and neurofibrillary tangles. Notably, rats with subchronic THP exposure showed vulnerability to neurofibrillary degeneration with misfolded tau protein aggregation compared with NS-treated, age-matched rats. Stereological quantification and immunoblotting revealed higher levels of phosphorylated tau AT8 and AT270 changes in THP-treated rats compared with age-matched controls. Long-term THP also accelerated a mature neurofibrillary pathology and neurodegeneration in neurons, and further, confirmed by Gallyas silver and FJB staining in the hippocampus of rat brain. Our study supports the hypothesis that long-term THP exposure affects the progression of neurodegeneration in these aging animals.
Despite the evidence described above, neurofibrillary degeneration coupled with an inflammatory response is not consistent with the cognitive behavioral performance. Our study shows that cognitive performance is disrupted as an acute impairment and is only present early on in THP treatment, likely as a consequence of cholinergic signaling blockage because of AC activity. Notably, this behavioral deficit did not continue or exacerbate upon repeated behavioral tests with subsequent THP treatment. Indeed, performances in the MWM and open field tests suggest that chronic THP exposure could lead to adaptation alteration or may be a compensatory mechanism to support cognitive performance, even with loss of neuronal activity. Accordingly, our study supports the hypothesis that THP impacts on the pathogenesis of neurodegeneration in laboratory animals. However, the neuropathological alterations related to these adaptations have yet to be confirmed. Additionally, our current study indicated THP-induced neuroimmune/neuroinflammatory dysfunction may be an event leading to earlier AD development. The neurodegenerative neurofibrillary pathogenesis that results from THP administration might be an earlier event according to the performance in behavioral tests. Moreover, there has been no systematic confirmation that acute or chronic prescription of such medications leads to transient or permanent adverse cognitive outcomes. Several gaps remain in the existing literature on the previously described association between AC use and clinical cognitive function. The mechanisms of action of medications determined in previous studies are inconsistent, making generation of a comprehensive, clinically useful list of AC medications impractical based on those data sets [
23,
24]. Thus, understanding such processes is vital because most drug therapies involve chronic AC administration. Our current study is the first to comprehensively measure the cognitive impact of long-term AC exposure on AD in aged laboratory animals. Neuroimmune/neuroinflammatory dysfunction is presented as one of the key differences between THP- and age-matched, NS-treated aging rats, accompanying deleterious neurodegenerative progression. AD is the most common form of dementia in the older population and is characterized by progressive neurodegeneration of the CNS. While the precise etiology of this disease still remains unknown, it is believed that intracellular accumulation of hyperphosphorylated tau (which forms neurofibrillary tangles) and deposition of extracellular filaments play a critical role in neurodegeneration. Accordingly, tau pathologies are evident in AD [
21,
25,
26]. From a molecular perspective, AD is a multifactorial disorder involving the association of genetic and environmental factors. Onset and progression of AD may be influenced by several risk factors such as hypertension, metabolic disorders, and/or inflammatory status [
27‐
29]. Owing to the limitations of our study, we cannot clarify the nature of interactions among THP, CNS inflammatory compartments, and neuronal neurodegeneration in this model. Future studies are necessary to determine how THP induces switching of systemic inflammatory events and progression of neurodegenerative pathology. Furthermore, additional studies examining the mechanism by which THP exerts its effects in more commonly used AD models should be performed. Nonetheless, our current results, together with the literature, provide clear implications for the rational use of ACs in older patients.
Abbreviations
AC, anticholinergic; AD, Alzheimer’s disease; CNS, central nervous system; FJB, Fluoro-Jade B; Iba1, ionized calcium binding adaptor molecule 1; IHC, immunohistochemical; IP, intraperitoneal; MHC, major histocompatibility complex; MWM, Morris water maze; NS, normal saline; PBS, phosphate-buffered saline; PD, Parkinson’s disease; qRT-PCR, quantitative real-time reverse transcription-polymerase chain reaction; SD, Sprague-Dawley rats; TBT, Tris-buffered saline; THP, trihexyphenidyl