Age-dependent impact of streptozotocin on metabolic endpoints and Alzheimer's disease pathologies in 3xTg-AD mice.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disease with a complex origin, thought to involve a combination of genetic, biological and environmental factors. Insulin dysfunction has emerged as a potential factor contributing to AD pathogenesis, particularly in individuals with diabetes, and among those with insulin deficiency or undergoing insulin therapy. The intraperitoneal administration of streptozotocin (STZ) is widely used in rodent models to explore the impact of insulin deficiency on AD pathology, although prior research predominantly focused on young animals, with no comparative analysis across different age groups. Our study aimed to fill this gap by analyzing the impact of insulin dysfunction in 7 and 23 months 3xTg-AD mice, that exhibit both amyloid and tau pathologies. Our objective was to elucidate the age-specific consequences of insulin deficiency on AD pathology. STZ administration led to insulin deficiency in the younger mice, resulting in an increase in cortical amyloid-ß (Aß) and tau aggregation, while tau phosphorylation was not significantly affected. Conversely, older mice displayed an unexpected resilience to the peripheral metabolic impact of STZ, while exhibiting an increase in both tau phosphorylation and aggregation without significantly affecting amyloid pathology. These changes were paralleled with alterations in signaling pathways involving tau kinases and phosphatases. Several markers of blood-brain barrier (BBB) integrity declined with age in 3xTg-AD mice, which might have facilitated a direct neurotoxic effect of STZ in older mice. Overall, our research confirms the influence of insulin signaling dysfunction on AD pathology, but also advises careful interpretation of data related to STZ-induced effects in older animals.

APOE Antibody Inhibits Aß-Associated Tau Seeding and Spreading in a Mouse Model.

APOE is the strongest genetic factor for late-onset Alzheimer's disease (AD). A specific conformation of the ApoE protein is present in amyloid-ß (Aß) containing plaques. Immunotherapy targeting ApoE in plaques reduces brain Aß deposits in mice. Here, we evaluated the effects of the anti-human APOE antibody HAE-4 on amyloid plaques, Aß-mediated tau seeding and spreading, and neuritic dystrophy in the 5XFAD amyloid mice expressing human ApoE4. HAE-4 reduced Aß plaques as well as Aß-driven tau seeding/spreading and neuritic dystrophy. These results demonstrate that HAE-4 may provide therapeutic effects on amyloid removal and Aß driven downstream consequences such as tauopathy. ANN NEUROL 2022;91:847-852.

Tau, Diabetes and Insulin.

Tau protein which was discovered in 1975 [310] became of great interest when it was identified as the main component of neurofibrillary tangles (NFT), a pathological feature in the brain of patients with Alzheimer's disease (AD) [39, 110, 232]. Tau protein is expressed mainly in the brain as six isoforms generated by alternative splicing [46, 97]. Tau is a microtubule associated proteins (MAPs) and plays a role in microtubules assembly and stability, as well as diverse cellular processes such as cell morphogenesis, cell division, and intracellular trafficking [49]. Additionally, Tau is involved in much larger neuronal functions particularly at the level of synapses and nuclei [11, 133, 280]. Tau is also physiologically released by neurons [233] even if the natural function of extracellular Tau remains to be uncovered (see other chapters of the present book).

Mutual Relationship between Tau and Central Insulin Signalling: Consequences for AD and Tauopathies?

Alzheimer disease (AD) is a progressive neurodegenerative disorder mainly characterized by cognitive deficits and neuropathological changes such as Tau lesions and amyloid plaques, but also associated with non-cognitive symptomatology. Metabolic and neuroendocrine abnormalities, such as alterations in body weight, brain insulin impairments, and lower brain glucose metabolism, which often precede clinical diagnosis, have been extensively reported in AD patients. However, the origin of these symptoms and their relation to pathology and cognitive impairments remain misunderstood. Insulin is a hormone involved in the control of energy homeostasis both peripherally and centrally, and insulin-resistant state has been linked to increased risk of dementia. It is now well established that insulin resistance can exacerbate Tau lesions, mainly by disrupting the balance between Tau kinases and phosphatases. On the other hand, the emerging literature indicates that Tau protein can also modulate insulin signalling in the brain, thus creating a detrimental vicious circle. The following review will highlight our current understanding of the role of insulin in the brain and its relation to Tau protein in the context of AD and tauopathies. Considering that insulin signalling is prone to be pharmacologically targeted at multiple levels, it constitutes an appealing approach to improve both insulin brain sensitivity and mitigate brain pathology with expected positive outcome in terms of cognition.

Tau hyperphosphorylation in the brain of ob/ob mice is due to hypothermia: Importance of thermoregulation in linking diabetes and Alzheimer's disease.

Over the last few decades, there has been a significant increase in epidemiological studies suggesting that type 2 diabetes (T2DM) is linked to a higher risk of Alzheimer's disease (AD). However, how T2DM affects AD pathology, such as tau hyperphosphorylation, is not well understood. In this study, we investigated the impact of T2DM on tau phosphorylation in ob/ob mice, a spontaneous genetic model of T2DM. Tau phosphorylation at the AT8 epitope was slightly elevated in 4-week-old ob/ob mice while 26-week-old ob/ob mice exhibited tau hyperphosphorylation at multiple tau phospho-epitopes (Tau1, CP13, AT8, AT180, PHF1). We then examined the mechanism of tau hyperphosphorylation and demonstrated that it is mostly due to hypothermia, as ob/ob mice were hypothermic and normothermia restored tau phosphorylation to control levels. As caffeine has been shown to be beneficial for diabetes, obesity and tau phosphorylation, we, therefore, used it as therapeutic treatment. Unexpectedly, chronic caffeine intake exacerbated tau hyperphosphorylation by promoting deeper hypothermia. Our data indicate that tau hyperphosphorylation is predominately due to hypothermia consequent to impaired thermoregulation in ob/ob mice. This study establishes a novel link between diabetes and AD, and reinforces the importance of recording body temperature to better assess the relationship between diabetes and AD.

Tau hyperphosphorylation and deregulation of calcineurin in mouse models of Huntington's disease.

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by polyglutamine expansions in the amino-terminal region of the huntingtin (Htt) protein. At the cellular level, neuronal death is accompanied by the proteolytic cleavage, misfolding and aggregation of huntingtin. Abnormal hyperphosphorylation of tau protein is a characteristic feature of a class of neurodegenerative diseases called tauopathies. As a number of studies have reported tau pathology in HD patients, we investigated whether HD pathology may promote tau hyperphosphorylation and if so tackle some of its underlying mechanisms. For that purpose, we used the R6/2 mouse, a well-characterized model of HD, and analyzed tau phosphorylation before and after the onset of HD-like symptoms. We found a significant increase in tau hyperphosphorylation at the PHF-1 epitope in pre-symptomatic R6/2 mice, whereas symptomatic mice displayed tau hyperphosphorylation at multiple tau phosphoepitopes (AT8, CP13, PT205 and PHF-1). There was no activation of major tau kinases that could explain this observation. However, when we examined tau phosphatases, we found that calcineurin/PP2B was downregulated by 30% in pre-symptomatic and 50% in symptomatic R6/2 mice, respectively. We observed similar changes in tau phosphorylation and calcineurin expression in Q175 mice, another HD model. Calcineurin was also reduced in Q111 compared with Q7 cells. Finally, pharmacological or genetic inhibition of endogenous calcineurin was sufficient to promote tau hyperphosphorylation in neuronal cells. Taken together, our data suggest that mutant huntingtin can induce abnormal tau hyperphosphorylation in vivo, via the deregulation of calcineurin.

Is Huntington's disease a tauopathy?

Tauopathies are a subclass of neurodegenerative diseases typified by the deposition of abnormal microtubule-associated tau protein within the cerebral tissue. Alzheimer's disease, progressive supranuclear palsy, chronic traumatic encephalopathy and some fronto-temporal dementias are examples of the extended family of tauopathies. In the last decades, intermittent reports of cerebral tau pathology in individuals afflicted with Huntington's disease-an autosomal dominant neurodegenerative disorder that manifests by severe motor, cognitive and psychiatric problems in adulthood-have also begun to surface. These observations remained anecdotal until recently when a series of publications brought forward compelling evidence that this monogenic disorder may, too, be a tauopathy. Collectively, these studies reported that: (i) patients with Huntington's disease present aggregated tau inclusions within various structures of the brain; (ii) tau haplotype influences the cognitive function of Huntington's disease patients; and (iii) that the genetic product of the disease, the mutant huntingtin protein, could alter tau splicing, phosphorylation, oligomerization and subcellular localization. Here, we review the past and current evidence in favour of the postulate that Huntington's disease is a new member of the family of tauopathies.

Hypothermia mediates age-dependent increase of tau phosphorylation in db/db mice.

Accumulating evidence from epidemiological studies suggest that type 2 diabetes is linked to an increased risk of Alzheimer's disease (AD). However, the consequences of type 2 diabetes on AD pathologies, such as tau hyperphosphorylation, are not well understood. Here, we evaluated the impact of type 2 diabetes on tau phosphorylation in db/db diabetic mice aged 4 and 26weeks. We found increased tau phosphorylation at the CP13 epitope correlating with a deregulation of c-Jun. N-terminal kinase (JNK) and Protein Phosphatase 2A (PP2A) in 4-week-old db/db mice. 26-week-old db/db mice displayed tau hyperphosphorylation at multiple epitopes (CP13, AT8, PHF-1), but no obvious change in kinases or phosphatases, no cleavage of tau, and no deregulation of central insulin signaling pathways. In contrast to younger animals, 26-week-old db/db mice were hypothermic and restoration of normothermia rescued phosphorylation at most epitopes. Our results suggest that, at early stages of type 2 diabetes, changes in tau phosphorylation may be due to deregulation of JNK and PP2A, while at later stages hyperphosphorylation is mostly a consequence of hypothermia. These results provide a novel link between diabetes and tau pathology, and underlie the importance of recording body temperature to better understand the relationship between diabetes and AD.

Parenchymal border macrophages regulate tau pathology and tau-mediated neurodegeneration.

Parenchymal border macrophages (PBMs) reside close to the central nervous system parenchyma and regulate CSF flow dynamics. We recently demonstrated that PBMs provide a clearance pathway for amyloid-ß peptide, which accumulates in the brain in Alzheimer's disease (AD). Given the emerging role for PBMs in AD, we explored how tau pathology affects the CSF flow and the PBM populations in the PS19 mouse model of tau pathology. We demonstrated a reduction of CSF flow, and an increase in an MHCII+PBM subpopulation in PS19 mice compared with WT littermates. Consequently, we asked whether PBM dysfunction could exacerbate tau pathology and tau-mediated neurodegeneration. Pharmacological depletion of PBMs in PS19 mice led to an increase in tau pathology and tau-dependent neurodegeneration, which was independent of gliosis or aquaporin-4 depolarization, essential for the CSF-ISF exchange. Together, our results identify PBMs as novel cellular regulators of tau pathology and tau-mediated neurodegeneration.

Astrocytic APOE4 removal confers cerebrovascular protection despite increased cerebral amyloid angiopathy.

BACKGROUND: Alzheimer Disease (AD) and cerebral amyloid angiopathy (CAA) are both characterized by amyloid-ß (Aß) accumulation in the brain, although Aß deposits mostly in the brain parenchyma in AD and in the cerebrovasculature in CAA. The presence of CAA can exacerbate clinical outcomes of AD patients by promoting spontaneous intracerebral hemorrhage and ischemia leading to CAA-associated cognitive decline. Genetically, AD and CAA share the ε4 allele of the apolipoprotein E (APOE) gene as the strongest genetic risk factor. Although tremendous efforts have focused on uncovering the role of APOE4 on parenchymal plaque pathogenesis in AD, mechanistic studies investigating the role of APOE4 on CAA are still lacking. Here, we addressed whether abolishing APOE4 generated by astrocytes, the major producers of APOE, is sufficient to ameliorate CAA and CAA-associated vessel damage. METHODS: We generated transgenic mice that deposited both CAA and plaques in which APOE4 expression can be selectively suppressed in astrocytes. At 2-months-of-age, a timepoint preceding CAA and plaque formation, APOE4 was removed from astrocytes of 5XFAD APOE4 knock-in mice. Mice were assessed at 10-months-of-age for Aß plaque and CAA pathology, gliosis, and vascular integrity. RESULTS: Reducing the levels of APOE4 in astrocytes shifted the deposition of fibrillar Aß from the brain parenchyma to the cerebrovasculature. However, despite increased CAA, astrocytic APOE4 removal reduced overall Aß-mediated gliosis and also led to increased cerebrovascular integrity and function in vessels containing CAA. CONCLUSION: In a mouse model of CAA, the reduction of  APOE4 derived specifically from astrocytes, despite increased fibrillar Aß deposition in the vasculature, is sufficient to reduce Aß-mediated gliosis and cerebrovascular dysfunction.

TREM2-independent microgliosis promotes tau-mediated neurodegeneration in the presence of ApoE4.

In addition to tau and Aß pathologies, inflammation plays an important role in Alzheimer's disease (AD). Variants in APOE and TREM2 increase AD risk. ApoE4 exacerbates tau-linked neurodegeneration and inflammation in P301S tau mice and removal of microglia blocks tau-dependent neurodegeneration. Microglia adopt a heterogeneous population of transcriptomic states in response to pathology, at least some of which are dependent on TREM2. Previously, we reported that knockout (KO) of TREM2 attenuated neurodegeneration in P301S mice that express mouse Apoe. Because of the possible common pathway of ApoE and TREM2 in AD, we tested whether TREM2 KO (T2KO) would block neurodegeneration in P301S Tau mice expressing ApoE4 (TE4), similar to that observed with microglial depletion. Surprisingly, we observed exacerbated neurodegeneration and tau pathology in TE4-T2KO versus TE4 mice, despite decreased TREM2-dependent microgliosis. Our results suggest that tau pathology-dependent microgliosis, that is, TREM2-independent microgliosis, facilitates tau-mediated neurodegeneration in the presence of ApoE4.

Sleep deprivation exacerbates microglial reactivity and Aß deposition in a TREM2-dependent manner in mice.

Sleep loss is associated with cognitive decline in the aging population and is a risk factor for Alzheimer's disease (AD). Considering the crucial role of immunomodulating genes such as that encoding the triggering receptor expressed on myeloid cells type 2 (TREM2) in removing pathogenic amyloid-ß (Aß) plaques and regulating neurodegeneration in the brain, our aim was to investigate whether and how sleep loss influences microglial function in mice. We chronically sleep-deprived wild-type mice and the 5xFAD mouse model of cerebral amyloidosis, expressing either the humanized TREM2 common variant, the loss-of-function R47H AD-associated risk variant, or without TREM2 expression. Sleep deprivation not only enhanced TREM2-dependent Aß plaque deposition compared with 5xFAD mice with normal sleeping patterns but also induced microglial reactivity that was independent of the presence of parenchymal Aß plaques. We investigated lysosomal morphology using transmission electron microscopy and found abnormalities particularly in mice without Aß plaques and also observed lysosomal maturation impairments in a TREM2-dependent manner in both microglia and neurons, suggesting that changes in sleep modified neuro-immune cross-talk. Unbiased transcriptome and proteome profiling provided mechanistic insights into functional pathways triggered by sleep deprivation that were unique to TREM2 and Aß pathology and that converged on metabolic dyshomeostasis. Our findings highlight that sleep deprivation directly affects microglial reactivity, for which TREM2 is required, by altering the metabolic ability to cope with the energy demands of prolonged wakefulness, leading to further Aß deposition, and underlines the importance of sleep modulation as a promising future therapeutic approach.

Targeting pre-synaptic tau accumulation: a new strategy to counteract tau-mediated synaptic loss and memory deficits.

Synaptic tau accumulation is believed to promote synaptic loss, which contributes to cognitive deficits in Alzheimer's disease and tauopathies. In this issue of Neuron, Largo-Barrientos et al. report that synaptic loss can be mitigated by lowering Synaptogyrin-3, a known mediator of tau binding to synaptic vesicles.

Activated microglia mitigate Aß-associated tau seeding and spreading.

In Alzheimer's disease (AD) models, AD risk variants in the microglial-expressed TREM2 gene decrease Aß plaque-associated microgliosis and increase neuritic dystrophy as well as plaque-associated seeding and spreading of tau aggregates. Whether this Aß-enhanced tau seeding/spreading is due to loss of microglial function or a toxic gain of function in TREM2-deficient microglia is unclear. Depletion of microglia in mice with established brain amyloid has no effect on amyloid but results in less spine and neuronal loss. Microglial repopulation in aged mice improved cognitive and neuronal deficits. In the context of AD pathology, we asked whether microglial removal and repopulation decreased Aß-driven tau seeding and spreading. We show that both TREM2KO and microglial ablation dramatically enhance tau seeding and spreading around plaques. Interestingly, although repopulated microglia clustered around plaques, they had a reduction in disease-associated microglia (DAM) gene expression and elevated tau seeding/spreading. Together, these data suggest that TREM2-dependent activation of the DAM phenotype is essential in delaying Aß-induced pathological tau propagation.

Selective removal of astrocytic APOE4 strongly protects against tau-mediated neurodegeneration and decreases synaptic phagocytosis by microglia.

The apolipoprotein E (APOE) gene is the strongest genetic risk factor for Alzheimer's disease and directly influences tauopathy and tau-mediated neurodegeneration. ApoE4 has strong deleterious effects on both parameters. In the brain, apoE is produced and secreted primarily by astrocytes and by activated microglia. The cell-specific role of each form of apoE in the setting of neurodegeneration has not been determined. We generated P301S Tau/Aldh1l1-CreERT2/apoE3flox/flox or Tau/Aldh1l1-CreERT2/apoE4flox/flox mice. At 5.5 months of age, after the onset of tau pathology, we administered tamoxifen or vehicle and compared mice at 9.5 months of age. Removing astrocytic APOE4 markedly reduced tau-mediated neurodegeneration and decreased phosphorylated tau (pTau) pathology. Single-nucleus RNA sequencing analysis revealed striking gene expression changes in all cell types, with astrocytic APOE4 removal decreasing disease-associated gene signatures in neurons, oligodendrocytes, astrocytes, and microglia. Removal of astrocytic APOE4 decreased tau-induced synaptic loss and microglial phagocytosis of synaptic elements, suggesting a key role for astrocytic apoE in synaptic degeneration.

APOE immunotherapy reduces cerebral amyloid angiopathy and amyloid plaques while improving cerebrovascular function.

The ε4 allele of the apolipoprotein E (APOE) gene is the strongest genetic risk factor for late-onset Alzheimer's disease (AD) and greatly influences the development of amyloid-ß (Aß) pathology. Our current study investigated the potential therapeutic effects of the anti-human APOE antibody HAE-4, which selectively recognizes human APOE that is co-deposited with Aß in cerebral amyloid angiopathy (CAA) and parenchymal amyloid pathology. In addition, we tested whether HAE-4 provoked brain hemorrhages, a component of amyloid-related imaging abnormalities (ARIA). ARIA is an adverse effect secondary to treatment with anti-Aß antibodies that can occur in blood vessels with CAA. We used 5XFAD mice expressing human APOE4 +/+ (5XE4) that have prominent CAA and parenchymal plaque pathology to assess the efficacy of HAE-4 compared to an Aß antibody that removes parenchymal Aß but increases ARIA in humans. In chronically treated 5XE4 mice, HAE-4 reduced Aß deposition including CAA compared to a control antibody, whereas the anti-Aß antibody had no effect on CAA. Furthermore, the anti-Aß antibody exacerbated microhemorrhage severity, which highly correlated with reactive astrocytes surrounding CAA. In contrast, HAE-4 did not stimulate microhemorrhages and instead rescued CAA-induced cerebrovascular dysfunction in leptomeningeal arteries in vivo. HAE-4 not only reduced amyloid but also dampened reactive microglial, astrocytic, and proinflammatory-associated genes in the cortex. These results suggest that targeting APOE in the core of both CAA and plaques could ameliorate amyloid pathology while protecting cerebrovascular integrity and function.

Circadian and sleep/wake-dependent variations in tau phosphorylation are driven by temperature.

STUDY OBJECTIVES: Aggregates of hyperphosphorylated tau protein are a hallmark of Alzheimer's disease (AD) and other tauopathies. Sleep disturbances are common in AD patients, and insufficient sleep may be a risk factor for AD. Recent evidence suggests that tau phosphorylation is dysregulated by sleep disturbances in mice. However, the physiological regulation of tau phosphorylation during the sleep-wake cycle is currently unknown. We thus aimed to determine whether tau phosphorylation is regulated by circadian rhythms, inherently linked to the sleep-wake cycle. METHODS: To answer these questions, we analyzed by Western blotting tau protein and associated kinases and phosphatases in the brains of awake, sleeping, and sleep-deprived B6 mice. We also recorded their temperature. RESULTS: We found that tau phosphorylation undergoes sleep-driven circadian variations as it is hyperphosphorylated during sleep but not during acute sleep deprivation. Moreover, we demonstrate that the mechanism behind these changes involves temperature, as tau phosphorylation was inversely correlated with circadian- and sleep deprivation-induced variations in body temperature, and prevented by housing the animals at a warmer temperature. Notably, similar changes in tau phosphorylation were reproduced in neuronal cells exposed to temperatures recorded during the sleep-wake cycle. Our results also suggest that inhibition of protein phosphatase 2A (PP2A) may explain the hyperphosphorylation of tau during sleep-induced hypothermia. CONCLUSION: Taken together, our results demonstrate that tau phosphorylation follows a circadian rhythm driven mostly by body temperature and sleep, and provide the physiological basis for further understanding how sleep deregulation can affect tau and ultimately AD pathology.

SEQUIN Multiscale Imaging of Mammalian Central Synapses Reveals Loss of Synaptic Connectivity Resulting from Diffuse Traumatic Brain Injury.

The brain's complex microconnectivity underlies its computational abilities and vulnerability to injury and disease. It has been challenging to illuminate the features of this synaptic network due to the small size and dense packing of its elements. Here, we describe a rapid, accessible super-resolution imaging and analysis workflow-SEQUIN-that quantifies central synapses in human tissue and animal models, characterizes their nanostructural and molecular features, and enables volumetric imaging of mesoscale synaptic networks without the production of large histological arrays. Using SEQUIN, we identify cortical synapse loss resulting from diffuse traumatic brain injury, a highly prevalent connectional disorder. Similar synapse loss is observed in three murine models of Alzheimer-related neurodegeneration, where SEQUIN mesoscale mapping identifies regional synaptic vulnerability. These results establish an easily implemented and robust nano-to-mesoscale synapse quantification and characterization method. They furthermore identify a shared mechanism-synaptopathy-between Alzheimer neurodegeneration and its best-established epigenetic risk factor, brain trauma.

Impact of TREM2R47H variant on tau pathology-induced gliosis and neurodegeneration.

Alzheimer's disease (AD) is characterized by plaques containing amyloid-ß (Aß) and neurofibrillary tangles composed of aggregated, hyperphosphorylated tau. Beyond tau and Aß, evidence suggests that microglia play an important role in AD pathogenesis. Rare variants in the microglia-expressed triggering receptor expressed on myeloid cells 2 (TREM2) gene increase AD risk 2- to 4-fold. It is likely that these TREM2 variants increase AD risk by decreasing the response of microglia to Aß and its local toxicity. However, neocortical Aß pathology occurs many years before neocortical tau pathology in AD. Thus, it will be important to understand the role of TREM2 in the context of tauopathy. We investigated the impact of the AD-associated TREM2 variant (R47H) on tau-mediated neuropathology in the PS19 mouse model of tauopathy. We assessed PS19 mice expressing human TREM2CV (common variant) or human TREM2R47H. PS19-TREM2R47H mice had significantly attenuated brain atrophy and synapse loss versus PS19-TREM2CV mice. Gene expression analyses and CD68 immunostaining revealed attenuated microglial reactivity in PS19-TREM2R47H versus PS19-TREM2CV mice. There was also a decrease in phagocytosis of postsynaptic elements by microglia expressing TREM2R47H in the PS19 mice and in human AD brains. These findings suggest that impaired TREM2 signaling reduces microglia-mediated neurodegeneration in the setting of tauopathy.

Administration of the benzodiazepine midazolam increases tau phosphorylation in the mouse brain.

Preclinical studies have shown that anesthesia might accelerate the clinical progression of Alzheimer's disease (AD) and can have an impact on tau pathology, a hallmark of AD. Although benzodiazepines have been suggested to increase the risk of incident dementia, their impact on tau pathology in vivo is unknown. We thus examined the impact of midazolam, a benzodiazepine that is often administered perioperatively as an anxiolytic, on tau hyperphosphorylation in nontransgenic and in hTau mice, the latter a model of AD-like tau pathology. The acute administration of midazolam in C57BL/6 mice was associated with downregulation of protein phosphatase-1 and a significant and persistent increase in brain tau phosphorylation. In hTau mice, tau hyperphosphorylation was also observed; however, midazolam was neither associated with proaggregant changes nor spatial reference memory impairment. In C57BL/6 mice, chronic midazolam administration immediately increased hippocampal tau phosphorylation, and this effect was more pronounced in older mice. Interestingly, in young C57BL/6 mice, chronic midazolam administration induced hippocampal tau hyperphosphorylation, which persisted for 1 week. In hTau mice, chronic midazolam administration increased hippocampal tau phosphorylation and, although this was not associated with proaggregant changes, this correlated with a decreased capacity of tau to bind to preassembled microtubules. These findings suggest that midazolam can induce significant tau hyperphosphorylation in vivo, which persists well beyond recovery from its sedative effects. Moreover, it can disrupt one of tau's critical functions. Hence, future studies should focus on the impact of more prolonged or repeated benzodiazepine exposure on tau pathology and cognitive decline.