Applications of CRISPR-Cas9 in Alzheimer's Disease and Related Disorders.

Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease represent some of the most prevalent neurodegenerative disorders afflicting millions of people worldwide. Unfortunately, there is a lack of efficacious treatments to cure or stop the progression of these disorders. While the causes of such a lack of therapies can be attributed to various reasons, the disappointing results of recent clinical trials suggest the need for novel and innovative approaches. Since its discovery, there has been a growing excitement around the potential for CRISPR-Cas9 mediated gene editing to identify novel mechanistic insights into disease pathogenesis and to mediate accurate gene therapy. To this end, the literature is rich with experiments aimed at generating novel models of these disorders and offering proof-of-concept studies in preclinical animal models validating the great potential and versatility of this gene-editing system. In this review, we provide an overview of how the CRISPR-Cas9 systems have been used in these neurodegenerative disorders.

Maternal choline supplementation ameliorates Alzheimer's disease pathology by reducing brain homocysteine levels across multiple generations.

The lack of effective treatments for Alzheimer's disease (AD) is alarming, considering the number of people currently affected by this disorder and the projected increase over the next few decades. Elevated homocysteine (Hcy) levels double the risk of developing AD. Choline, a primary dietary source of methyl groups, converts Hcy to methionine and reduces age-dependent cognitive decline. Here, we tested the transgenerational benefits of maternal choline supplementation (ChS; 5.0 g/kg choline chloride) in two generations (Gen) of APP/PS1 mice. We first exposed 2.5-month-old mice to the ChS diet and allowed them to breed with each other to generate Gen-1 mice. Gen-1 mice were exposed to the ChS diet only during gestation and lactation; once weaned at postnatal day 21, Gen-1 mice were then kept on the control diet for the remainder of their life. We also bred a subset of Gen-1 mice to each other and obtained Gen-2 mice; these mice were never exposed to ChS. We found that ChS reduced Aß load and microglia activation, and improved cognitive deficits in old Gen-1 and Gen-2 APP/PS1 mice. Mechanistically, these changes were linked to a reduction in brain Hcy levels in both generations. Further, RNA-Seq data from APP/PS1 hippocampal tissue revealed that ChS significantly changed the expression of 27 genes. These genes were enriched for inflammation, histone modifications, and neuronal death functional classes. Our results are the first to demonstrate a transgenerational benefit of ChS and suggest that modifying the maternal diet with additional choline reduces AD pathology across multiple generations.

Genetic reduction of Nrf2 exacerbates cognitive deficits in a mouse model of Alzheimer's disease.

Aging is the major risk factor for several neurodegenerative diseases, including Alzheimer's disease (AD). However, the mechanisms by which aging contributes to neurodegeneration remain elusive. The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a transcription factor that regulates expression of a vast number of genes by binding to the antioxidant response element. Nrf2 levels decrease as a function of age, and reduced Nrf2 levels have been reported in postmortem human brains and animal models of AD. Nevertheless, it is still unknown whether Nrf2 plays a role in the cognitive deficits associated with AD. To address this question, we used a genetic approach to remove the Nrf2 gene from APP/PS1 mice, a widely used animal model of AD. We found that the lack of Nrf2 significantly exacerbates cognitive deficits in APP/PS1, without altering gross motor function. Specifically, we found an exacerbation of deficits in spatial learning and memory, as well as in working and associative memory. Different brain regions control these behavioral tests, indicating that the lack of Nrf2 has a global effect on brain function. The changes in cognition were linked to an increase in Aß and interferon-gamma (IFNγ) levels, and microgliosis. The changes in IFNγ levels are noteworthy as previously published evidence indicates that IFNγ can increase microglia activation and induce Aß production. Our data suggest a clear link between Nrf2 and AD-mediated cognitive decline and further strengthen the connection between Nrf2 and AD.

Reduced protein turnover mediates functional deficits in transgenic mice expressing the 25 kDa C-terminal fragment of TDP-43.

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP) are two neurodegenerative disorders characterized by the accumulation of TDP-43. TDP-43 is proteolitically cleaved to generate two major C-terminal fragments of 35 and 25 kDa. The latter, known as TDP-25, is a consistent feature of FTLD-TDP and ALS; however, little is known about its role in disease pathogenesis. We have previously developed transgenic mice overexpressing low levels of TDP-25 (TgTDP-25(+/0)), which at 6 months of age show mild cognitive impairments and no motor deficits. To better understand the role of TDP-25 in the pathogenesis of ALS and FTLD-TDP, we generated TDP-25 homozygous mice (TgTDP-25(+/+)), thereby further increasing TDP-25 expression. We found a gene-dosage effect on cognitive and motor function at 15 months of age, as the TgTDP-25(+/+) showed more severe spatial and working memory deficits as well as worse motor performance than TgTDP-25(+/0) mice. These behavioral deficits were associated with increased soluble levels of TDP-25 in the nucleus and cytosol. Notably, high TDP-25 levels were also linked to reduced autophagy induction and proteasome function, two events that have been associated with both ALS and FTLD-TDP. In summary, we present strong in vivo evidence that high levels of TDP-25 are sufficient to cause behavioral deficits and reduce function of two of the major protein turnover systems: autophagy and proteasome. These mice represent a new tool to study the role of TDP-25 in the pathogenesis of ALS and FTLD-TDP.

Reducing Ribosomal Protein S6 Kinase 1 Expression Improves Spatial Memory and Synaptic Plasticity in a Mouse Model of Alzheimer's Disease.

Aging is the most important risk factor associated with Alzheimer's disease (AD); however, the molecular mechanisms linking aging to AD remain unclear. Suppression of the ribosomal protein S6 kinase 1 (S6K1) increases healthspan and lifespan in several organisms, from nematodes to mammals. Here we show that S6K1 expression is upregulated in the brains of AD patients. Using a mouse model of AD, we found that genetic reduction of S6K1 improved synaptic plasticity and spatial memory deficits, and reduced the accumulation of amyloid-ß and tau, the two neuropathological hallmarks of AD. Mechanistically, these changes were linked to reduced translation of tau and the ß-site amyloid precursor protein cleaving enzyme 1, a key enzyme in the generation of amyloid-ß. Our results implicate S6K1 dysregulation as a previously unidentified molecular mechanism underlying synaptic and memory deficits in AD. These findings further suggest that therapeutic manipulation of S6K1 could be a valid approach to mitigate AD pathology. SIGNIFICANCE STATEMENT: Aging is the most important risk factor for Alzheimer's disease (AD). However, little is known about how it contributes to AD pathogenesis. S6 kinase 1 (S6K1) is a protein kinase involved in regulation of protein translation. Reducing S6K1 activity increases lifespan and healthspan. We report the novel finding that reducing S6K1 activity in 3xTg-AD mice ameliorates synaptic and cognitive deficits. These improvement were associated with a reduction in amyloid-ß and tau pathology. Mechanistically, lowering S6K1 levels reduced translation of ß-site amyloid precursor protein cleaving enzyme 1 and tau, two key proteins involved in AD pathogenesis. These data suggest that S6K1 may represent a molecular link between aging and AD. Given that aging is the most important risk factor for most neurodegenerative diseases, our results may have far-reaching implications into other diseases.

Genetic reduction of mammalian target of rapamycin ameliorates Alzheimer's disease-like cognitive and pathological deficits by restoring hippocampal gene expression signature.

Elevated mammalian target of rapamycin (mTOR) signaling has been found in Alzheimer's disease (AD) patients and is linked to diabetes and aging, two known risk factors for AD. However, whether hyperactive mTOR plays a role in the cognitive deficits associated with AD remains elusive. Here, we genetically reduced mTOR signaling in the brains of Tg2576 mice, a widely used animal model of AD. We found that suppression of mTOR signaling reduced amyloid-ß deposits and rescued memory deficits. Mechanistically, the reduction in mTOR signaling led to an increase in autophagy induction and restored the hippocampal gene expression signature of the Tg2576 mice to wild-type levels. Our results implicate hyperactive mTOR signaling as a previous unidentified signaling pathway underlying gene-expression dysregulation and cognitive deficits in AD. Furthermore, hyperactive mTOR signaling may represent a molecular pathway by which aging contributes to the development of AD.

Glucocorticoids exacerbate cognitive deficits in TDP-25 transgenic mice via a glutathione-mediated mechanism: implications for aging, stress and TDP-43 proteinopathies.

The accumulation of TDP-43 (transactive response DNA-binding protein 43) and its 25 kDa C-terminal fragment (TDP-25) is a hallmark of several neurodegenerative disorders, including frontotemporal lobar degeneration (FTLD-TDP) and amyotrophic lateral sclerosis (ALS). The majority of FTLD-TDP cases are due to loss of function mutations in the gene encoding progranulin, a secreted growth factor. In ALS, specific mutations in the gene encoding TDP-43 have been linked to the disease pathogenesis. In both cases, however, the penetrance of the mutations greatly increases during aging, suggesting that other genetic or environmental factors may facilitate the development of the disease. Using transgenic mice that overexpress the 25 kDa C-terminal fragment of TDP-43, here we show that glucocorticoids, stress hormones known to increase the brain susceptibility to neurotoxic insults, increase the levels of soluble TDP-25 and exacerbate cognitive deficits, without altering full-length TDP-43 levels. Additionally, we show that the mechanism underlying the glucocorticoid-mediated increase in TDP-25 levels is coupled to changes in the glutathione redox state. Glutathione is an antioxidant involved in protecting cells from damage caused by reactive oxygen species; notably, alterations in the ratio of reduced to oxidized glutathione, which is the primary determinant of the cellular redox state, are associated with aging and neurodegeneration. We show that restoring the ratio of reduced to oxidized glutathione blocks the glucocorticoid effects on TDP-25. These data show that glucocorticoids potentiate the neurotoxic action of TDP-25 by increasing its levels and clearly indicate the role of cellular oxidative damage in this process.

Oxidative damage and amyloid-ß metabolism in brain regions of the longest-lived rodents.

Naked mole rats (NMRs) are the longest-lived rodents, with young individuals having high levels of Aß in their brains. The purpose of this study was twofold: to assess the distribution of Aß in key regions of NMR brains (cortex, hippocampus, cerebellum) and to understand whether the accumulation of Aß is due to enhanced production or decreased degradation. Recent evidence indicates that lipid peroxides directly participate in induction of cytoprotective proteins, such as heat shock proteins (Hsps), which play a central role in the cellular mechanisms of stress tolerance. Amyloid precursor protein processing, lipid peroxidation, Hsps, redox status, and protein degradation processes were therefore assessed in key NMR brain regions. NMR brains had high levels of lipid peroxidation compared with mice, and the NMR hippocampus had the highest levels of the most toxic moiety of Aß (soluble Aß1 - 42 ). This was due not to increased Aß production but rather to low antioxidant potential, which was associated with low induction of Hsp70 and heme oxygenase-1 as well as low ubiquitin-proteasome activity. NMRs may therefore serve as natural models for understanding the relationship between oxidative stress and Aß levels and its effects on the brain.

Epigenetic Changes in Alzheimer's Disease: DNA Methylation and Histone Modification.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder characterized by progressive cognitive decline and memory loss, imposing a significant burden on affected individuals and their families. Despite the recent promising progress in therapeutic approaches, more needs to be done to understand the intricate molecular mechanisms underlying the development and progression of AD. Growing evidence points to epigenetic changes as playing a pivotal role in the pathogenesis of the disease. The dynamic interplay between genetic and environmental factors influences the epigenetic landscape in AD, altering gene expression patterns associated with key pathological events associated with disease pathogenesis. To this end, epigenetic alterations not only impact the expression of genes implicated in AD pathogenesis but also contribute to the dysregulation of crucial cellular processes, including synaptic plasticity, neuroinflammation, and oxidative stress. Understanding the complex epigenetic mechanisms in AD provides new avenues for therapeutic interventions. This review comprehensively examines the role of DNA methylation and histone modifications in the context of AD. It aims to contribute to a deeper understanding of AD pathogenesis and facilitate the development of targeted therapeutic strategies.

Anti-Biofilm Strategies: A Focused Review on Innovative Approaches.

Biofilm (BF) can give rise to systemic infections, prolonged hospitalization times, and, in the worst case, death. This review aims to provide an overview of recent strategies for the prevention and destruction of pathogenic BFs. First, the main phases of the life cycle of BF and maturation will be described to identify potential targets for anti-BF approaches. Then, an approach acting on bacterial adhesion, quorum sensing (QS), and the extracellular polymeric substance (EPS) matrix will be introduced and discussed. Finally, bacteriophage-mediated strategies will be presented as innovative approaches against BF inhibition/destruction.

AAV-mediated upregulation of VDAC1 rescues the mitochondrial respiration and sirtuins expression in a SOD1 mouse model of inherited ALS.

Mitochondrial dysfunction represents one of the most common molecular hallmarks of both sporadic and familial forms of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder caused by the selective degeneration and death of motor neurons. The accumulation of misfolded proteins on and within mitochondria, as observed for SOD1 G93A mutant, correlates with a drastic reduction of mitochondrial respiration and the inhibition of metabolites exchanges, including ADP/ATP and NAD+/NADH, across the Voltage-Dependent Anion-selective Channel 1 (VDAC1), the most abundant channel protein of the outer mitochondrial membrane. Here, we show that the AAV-mediated upregulation of VDAC1 in the spinal cord of transgenic mice expressing SOD1 G93A completely rescues the mitochondrial respiratory profile. This correlates with the increased activity and levels of key regulators of mitochondrial functions and maintenance, namely the respiratory chain Complex I and the sirtuins (Sirt), especially Sirt3. Furthermore, the selective increase of these mitochondrial proteins is associated with an increase in Tom20 levels, the receptor subunit of the TOM complex. Overall, our results indicate that the overexpression of VDAC1 has beneficial effects on ALS-affected tissue by stabilizing the Complex I-Sirt3 axis.

Cognitive decline typical of frontotemporal lobar degeneration in transgenic mice expressing the 25-kDa C-terminal fragment of TDP-43.

Transactive response DNA-binding protein 43 (TDP-43) is the pathological signature protein in several neurodegenerative disorders, including the majority of frontotemporal lobar degeneration cases (FTLD-TDP), motor neuron disease, and amyotrophic lateral sclerosis. Pathological TDP-43 is mislocalized from its nuclear location to the cytoplasm, where it accumulates and is proteolytically cleaved to form C-terminal fragments. Although the 25-kDa C-terminal fragment of TDP-43 (TDP-25) accumulates in affected brain regions, its role in the disease pathogenesis remains elusive. To address this problem, we have generated a novel transgenic mouse that selectively expresses TDP-25 in neurons. We show that transgenic mice expressing TDP-25 develop cognitive deficits associated with the build-up of soluble TDP-25. These cognitive deficits are independent of TDP-43-positive inclusions and occur without overt neurodegeneration. Additionally, we show that the expression of TDP-25 is sufficient to alter the processing of endogenous full-length TDP-43. These studies represent the first in vivo demonstration of a pathological role for TDP-25 and strongly suggest that the onset of cognitive deficits in TDP-43 proteinopathies is independent of TDP-43 inclusions. These data provide a framework for understanding the molecular mechanisms underlying the onset of cognitive deficits in FTLD-TDP and other TDP-43 proteinopathies; thus, the TDP-25 transgenic mice represent a unique tool to reach this goal.

CBP gene transfer increases BDNF levels and ameliorates learning and memory deficits in a mouse model of Alzheimer's disease.

Cognitive dysfunction and memory loss are common features of Alzheimer's disease (AD). Abnormalities in the expression profile of immediate early genes that play a critical role in memory formation, such as the cAMP-response element binding protein (CREB), have been reported in the brains of AD patients. Here we show that amyloid-ß (Aß) accumulation, which plays a primary role in the cognitive deficits of AD, interferes with CREB activity. We further show that restoring CREB function via brain viral delivery of the CREB-binding protein (CBP) improves learning and memory deficits in an animal model of AD. Notably, such improvements occur without changes in Aß and tau pathology, and instead are linked to an increased level of brain-derived neurotrophic factor. The resulting data suggest that Aß-induced learning and memory deficits are mediated by alterations in CREB function, based on the finding that restoring CREB activity by directly modulating CBP levels in the brains of adult mice is sufficient to ameliorate learning and memory. Therefore, increasing CBP expression in adult brains may be a valid therapeutic approach not only for AD, but also for various brain disorders characterized by alterations in immediate early genes, further supporting the concept that viral vector delivery may be a viable therapeutic approach in neurodegenerative diseases.

ERK1/2-dependent TSPO overactivation associates with the loss of mitophagy and mitochondrial respiration in ALS.

Mitochondrial dysfunction and the loss of mitophagy, aimed at recycling irreversibly damaged organelles, contribute to the onset of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease affecting spinal cord motor neurons. In this work, we showed that the reduction of mitochondrial respiration, exactly oxygen flows linked to ATP production and maximal capacity, correlates with the appearance of the most common ALS motor symptoms in a transgenic mouse model expressing SOD1 G93A mutant. This is the result of the equal inhibition in the respiration linked to complex I and II of the electron transport chain, but not their protein levels. Since the overall mitochondrial mass was unvaried, we investigated the expression of the Translocator Protein (TSPO), a small mitochondrial protein whose overexpression was recently linked to the loss of mitophagy in a model of Parkinson's disease. Here we clearly showed that levels of TSPO are significantly increased in ALS mice. Mechanistically, this increase is linked to the overactivation of ERK1/2 pathway and correlates with a decrease in the expression of the mitophagy-related marker Atg12, indicating the occurrence of impairments in the activation of mitophagy. Overall, our work sets out TSPO as a key regulator of mitochondrial homeostasis in ALS.

The Role of the Transcription Factor Nrf2 in Alzheimer's Disease: Therapeutic Opportunities.

Alzheimer's disease (AD) is a common neurodegenerative disorder that affects the elderly. One of the key features of AD is the accumulation of reactive oxygen species (ROS), which leads to an overall increase in oxidative damage. The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a master regulator of the antioxidant response in cells. Under low ROS levels, Nrf2 is kept in the cytoplasm. However, an increase in ROS production leads to a translocation of Nrf2 into the nucleus, where it activates the transcription of several genes involved in the cells' antioxidant response. Additionally, Nrf2 activation increases autophagy function. However, in AD, the accumulation of Aß and tau reduces Nrf2 levels, decreasing the antioxidant response. The reduced Nrf2 levels contribute to the further accumulation of Aß and tau by impairing their autophagy-mediated turnover. In this review, we discuss the overwhelming evidence indicating that genetic or pharmacological activation of Nrf2 is as a potential approach to mitigate AD pathology.

Generation of a helper phage for the fluorescent detection of peptide-target interactions by dual-display phages.

Phage display is a molecular biology technique that allows the presentation of foreign peptides on the surface of bacteriophages. It is widely utilized for applications such as the discovery of biomarkers, the development of therapeutic antibodies, and the investigation of protein-protein interactions. When employing phages in diagnostic and therapeutic monitoring assays, it is essential to couple them with a detection system capable of revealing and quantifying the interaction between the peptide displayed on the phage capsid and the target of interest. This process is often technically challenging and costly. Here, we generated a fluorescent helper phage vector displaying sfGFP in-frame to the pIII of the capsid proteins. Further, we developed an exchangeable dual-display phage system by combining our newly developed fluorescent helper phage vector with a phagemid vector harboring the engineered pVIII with a peptide-probe. By doing so, the sfGFP and a peptide-probe are displayed on the same phage particle. Notably, our dual-display approach is highly flexible as it allows for easy exchange of the displayed peptide-probe on the pVIII to gain the desired selectivity, while maintaining the sfGFP gene, which allows easy visualization and quantification of the interaction peptide-probe. We anticipate that this system will reduce time and costs compared to the current phage-based detection systems.

Selective Noradrenaline Depletion in the Neocortex and Hippocampus Induces Working Memory Deficits and Regional Occurrence of Pathological Proteins.

Noradrenaline (NA) depletion occurs in Alzheimer's disease (AD); however, its relationship with the pathological expression of Tau and transactive response DNA-binding protein 43 (TDP-43), two major hallmarks of AD, remains elusive. Here, increasing doses of a selective noradrenergic immunotoxin were injected into developing rats to generate a model of mild or severe NA loss. At about 12 weeks post-lesion, dose-dependent working memory deficits were detected in these animals, associated with a marked increase in cortical and hippocampal levels of TDP-43 phosphorylated at Ser 409/410 and Tau phosphorylated at Thr 217. Notably, the total levels of both proteins were largely unaffected, suggesting a direct relationship between neocortical/hippocampal NA depletion and the phosphorylation of pathological Tau and TDP-43 proteins. As pTD43 is present in 23% of AD cases and pTau Thr217 has been detected in patients with mild cognitive impairment that eventually would develop into AD, improvement of noradrenergic function in AD might represent a viable therapeutic approach with disease-modifying potential.

Physiologic Response Evaluation of Human Foetal Osteoblast Cells within Engineered 3D-Printed Polylactic Acid Scaffolds.

Large bone defect treatments have always been one of the important challenges in clinical practice and created a huge demand for more efficacious regenerative approaches. The bone tissue engineering (BTE) approach offered a new alternative to conventional bone grafts, addressing all clinical needs. Over the past years, BTE research is focused on the study and realisation of new biomaterials, including 3D-printed supports to improve mechanical, structural and biological properties. Among these, polylactic acid (PLA) scaffolds have been considered the most promising biomaterials due to their good biocompatibility, non-toxic biodegradability and bioresorbability. In this work, we evaluated the physiological response of human foetal osteoblast cells (hFOB), in terms of cell proliferation and osteogenic differentiation, within oxygen plasma treated 3D-printed PLA scaffolds, obtained by fused deposition modelling (FDM). A mechanical simulation to predict their behaviour to traction, flexural or torque solicitations was performed. We found that: 1. hFOB cells adhere and grow on scaffold surfaces; 2. hFOB grown on oxygen plasma treated PLA scaffolds (PLA_PT) show an improvement of cell adhesion and proliferation, compared to not-plasma treated scaffolds (PLA_NT); 3. Over time, hFOB penetrate along strands, differentiate, and form a fibrous matrix, tissue-like; 4. 3D-printed PLA scaffolds have good mechanical behaviour in each analysed configuration. These findings suggest that 3D-printed PLA scaffolds could represent promising biomaterials for medical implantable devices in the orthopaedic field.

Naturally secreted amyloid-beta increases mammalian target of rapamycin (mTOR) activity via a PRAS40-mediated mechanism.

Reducing the mammalian target of rapamycin (mTOR) activity increases lifespan and health span in a variety of organisms. Alterations in protein homeostasis and mTOR activity and signaling have been reported in several neurodegenerative disorders, including Alzheimer disease (AD); however, the causes of such deregulations remain elusive. Here, we show that mTOR activity and signaling are increased in cell lines stably transfected with mutant amyloid precursor protein (APP) and in brains of 3xTg-AD mice, an animal model of AD. In addition, we show that in the 3xTg-AD mice, mTOR activity can be reduced to wild type levels by genetically preventing Aß accumulation. Similarly, intrahippocampal injections of an anti-Aß antibody reduced Aß levels and normalized mTOR activity, indicating that high Aß levels are necessary for mTOR hyperactivity in 3xTg-AD mice. We also show that the intrahippocampal injection of naturally secreted Aß is sufficient to increase mTOR signaling in the brains of wild type mice. The mechanism behind the Aß-induced mTOR hyperactivity is mediated by the proline-rich Akt substrate 40 (PRAS40) as we show that the activation of PRAS40 plays a key role in the Aß-induced mTOR hyperactivity. Taken together, our data show that Aß accumulation, which has been suggested to be the culprit of AD pathogenesis, causes mTOR hyperactivity by regulating PRAS40 phosphorylation. These data further indicate that the mTOR pathway is one of the pathways by which Aß exerts its toxicity and further support the idea that reducing mTOR signaling in AD may be a valid therapeutic approach.

Loss of muscarinic M1 receptor exacerbates Alzheimer's disease-like pathology and cognitive decline.

Alzheimer's disease (AD) is pathologically characterized by tau-laden neurofibrillary tangles and ß-amyloid deposits. Dysregulation of cholinergic neurotransmission has been implicated in AD pathogenesis, contributing to the associated memory impairments; yet, the exact mechanisms remain to be defined. Activating the muscarinic acetylcholine M(1) receptors (M(1)Rs) reduces AD-like pathological features and enhances cognition in AD transgenic models. To elucidate the molecular mechanisms by which M(1)Rs affect AD pathophysiological features, we crossed the 3xTgAD and transgenic mice expressing human Swedish, Dutch, and Iowa triple-mutant amyloid precursor protein (Tg-SwDI), two widely used animal models, with the M(1)R(-/-) mice. Our data show that M(1)R deletion in the 3xTgAD and Tg-SwDI mice exacerbates the cognitive impairment through mechanisms dependent on the transcriptional dysregulation of genes required for memory and through acceleration of AD-related synaptotoxicity. Ablating the M(1)R increased plaque and tangle levels in the brains of 3xTgAD mice and elevated cerebrovascular deposition of fibrillar Aß in Tg-SwDI mice. Notably, tau hyperphosphorylation and potentiation of amyloidogenic processing in the mice with AD lacking M(1)R were attributed to changes in the glycogen synthase kinase 3ß and protein kinase C activities. Finally, deleting the M(1)R increased the astrocytic and microglial response associated with Aß plaques. Our data highlight the significant role that disrupting the M(1)R plays in exacerbating AD-related cognitive decline and pathological features and provide critical preclinical evidence to justify further development and evaluation of selective M(1)R agonists for treating AD.