Alzheimer risk-increasing TREM2 variant causes aberrant cortical synapse density and promotes network hyperexcitability in mouse models.

The R47H variant of triggering receptor expressed on myeloid cells 2 (TREM2) increases the risk of Alzheimer's disease (AD). To investigate potential mechanisms, we analyzed knockin mice expressing human TREM2-R47H from one mutant mouse Trem2 allele. TREM2-R47H mice showed increased seizure activity in response to an acute excitotoxin challenge, compared to wildtype controls or knockin mice expressing the common variant of human TREM2. TREM2-R47H also increased spontaneous thalamocortical epileptiform activity in App knockin mice expressing amyloid precursor proteins bearing autosomal dominant AD mutations and a humanized amyloid-ß sequence. In mice with or without such App modifications, TREM2-R47H increased the density of putative synapses in cortical regions without amyloid plaques. TREM2-R47H did not affect synaptic density in hippocampal regions with or without plaques. We conclude that TREM2-R47H increases AD-related network hyperexcitability and that it may do so, at least in part, by causing an imbalance in synaptic densities across brain regions.

TAU ablation in excitatory neurons and postnatal TAU knockdown reduce epilepsy, SUDEP, and autism behaviors in a Dravet syndrome model.

Intracellular accumulation of TAU aggregates is a hallmark of several neurodegenerative diseases. However, global genetic reduction of TAU is beneficial also in models of other brain disorders that lack such TAU pathology, suggesting a pathogenic role of nonaggregated TAU. Here, conditional ablation of TAU in excitatory, but not inhibitory, neurons reduced epilepsy, sudden unexpected death in epilepsy, overactivation of the phosphoinositide 3-kinase-AKT-mammalian target of rapamycin pathway, brain overgrowth (megalencephaly), and autism-like behaviors in a mouse model of Dravet syndrome, a severe epileptic encephalopathy of early childhood. Furthermore, treatment with a TAU-lowering antisense oligonucleotide, initiated on postnatal day 10, had similar therapeutic effects in this mouse model. Our findings suggest that excitatory neurons are the critical cell type in which TAU has to be reduced to counteract brain dysfunctions associated with Dravet syndrome and that overall cerebral TAU reduction could have similar benefits, even when initiated postnatally.

Interdependence of neural network dysfunction and microglial alterations in Alzheimer's disease-related models.

Nonconvulsive epileptiform activity and microglial alterations have been detected in people with Alzheimer's disease (AD) and related mouse models. However, the relationship between these abnormalities remains to be elucidated. We suppressed epileptiform activity by treatment with the antiepileptic drug levetiracetam or by genetic ablation of tau and found that these interventions reversed or prevented aberrant microglial gene expression in brain tissues of aged human amyloid precursor protein transgenic mice, which simulate several key aspects of AD. The most robustly modulated genes included multiple factors previously implicated in AD pathogenesis, including TREM2, the hypofunction of which increases disease risk. Genetic reduction of TREM2 exacerbated epileptiform activity after mice were injected with kainate. We conclude that AD-related epileptiform activity markedly changes the molecular profile of microglia, inducing both maladaptive and adaptive alterations in their activities. Increased expression of TREM2 seems to support microglial activities that counteract this type of network dysfunction.

Tau reduction affects excitatory and inhibitory neurons differently, reduces excitation/inhibition ratios, and counteracts network hypersynchrony.

The protein tau has been implicated in many brain disorders. In animal models, tau reduction suppresses epileptogenesis of diverse causes and ameliorates synaptic and behavioral abnormalities in various conditions associated with excessive excitation-inhibition (E/I) ratios. However, the underlying mechanisms are unknown. Global genetic ablation of tau in mice reduces the action potential (AP) firing and E/I ratio of pyramidal cells in acute cortical slices without affecting the excitability of these cells. Tau ablation reduces the excitatory inputs to inhibitory neurons, increases the excitability of these cells, and structurally alters their axon initial segments (AISs). In primary neuronal cultures subjected to prolonged overstimulation, tau ablation diminishes the homeostatic response of AISs in inhibitory neurons, promotes inhibition, and suppresses hypersynchrony. Together, these differential alterations in excitatory and inhibitory neurons help explain how tau reduction prevents network hypersynchrony and counteracts brain disorders causing abnormally increased E/I ratios.

Phenotypic Differences between the Alzheimer's Disease-Related hAPP-J20 Model and Heterozygous Zbtb20 Knock-Out Mice.

Diverse gene products contribute to the pathogenesis of Alzheimer's disease (AD). Experimental models have helped elucidate their mechanisms and impact on brain functions. Human amyloid precursor protein (hAPP) transgenic mice from line J20 (hAPP-J20 mice) are widely used to simulate key aspects of AD. However, they also carry an insertional mutation in noncoding sequence of one Zbtb20 allele, a gene involved in neural development. We demonstrate that heterozygous hAPP-J20 mice have reduced Zbtb20 expression in some AD-relevant brain regions, but not others, and that Zbtb20 levels are higher in hAPP-J20 mice than heterozygous Zbtb20 knock-out (Zbtb20+/-) mice. Whereas hAPP-J20 mice have premature mortality, severe deficits in learning and memory, other behavioral alterations, and prominent nonconvulsive epileptiform activity, Zbtb20+/- mice do not. Thus, the insertional mutation in hAPP-J20 mice does not ablate the affected Zbtb20 allele and is unlikely to account for the AD-like phenotype of this model.

Behavioral and neural network abnormalities in human APP transgenic mice resemble those of App knock-in mice and are modulated by familial Alzheimer's disease mutations but not by inhibition of BACE1.

BACKGROUND: Alzheimer's disease (AD) is the most frequent and costly neurodegenerative disorder. Although diverse lines of evidence suggest that the amyloid precursor protein (APP) is involved in its causation, the precise mechanisms remain unknown and no treatments are available to prevent or halt the disease. A favorite hypothesis has been that APP contributes to AD pathogenesis through the cerebral accumulation of the amyloid-ß peptide (Aß), which is derived from APP through sequential proteolytic cleavage by BACE1 and γ-secretase. However, inhibitors of these enzymes have failed in clinical trials despite clear evidence for target engagement. METHODS: To further elucidate the roles of APP and its metabolites in AD pathogenesis, we analyzed transgenic mice overexpressing wildtype human APP (hAPP) or hAPP carrying mutations that cause autosomal dominant familial AD (FAD), as well as App knock-in mice that do not overexpress hAPP but have two mouse App alleles with FAD mutations and a humanized Aß sequence. RESULTS: Although these lines of mice had marked differences in cortical and hippocampal levels of APP, APP C-terminal fragments, soluble Aß, Aß oligomers and age-dependent amyloid deposition, they all developed cognitive deficits as well as non-convulsive epileptiform activity, a type of network dysfunction that also occurs in a substantive proportion of humans with AD. Pharmacological inhibition of BACE1 effectively reduced levels of amyloidogenic APP C-terminal fragments (C99), soluble Aß, Aß oligomers, and amyloid deposits in transgenic mice expressing FAD-mutant hAPP, but did not improve their network dysfunction and behavioral abnormalities, even when initiated at early stages before amyloid deposits were detectable. CONCLUSIONS: hAPP transgenic and App knock-in mice develop similar pathophysiological alterations. APP and its metabolites contribute to AD-related functional alterations through complex combinatorial mechanisms that may be difficult to block with BACE inhibitors and, possibly, also with other anti-Aß treatments.

A second X chromosome contributes to resilience in a mouse model of Alzheimer's disease.

A major sex difference in Alzheimer's disease (AD) is that men with the disease die earlier than do women. In aging and preclinical AD, men also show more cognitive deficits. Here, we show that the X chromosome affects AD-related vulnerability in mice expressing the human amyloid precursor protein (hAPP), a model of AD. XY-hAPP mice genetically modified to develop testicles or ovaries showed worse mortality and deficits than did XX-hAPP mice with either gonad, indicating a sex chromosome effect. To dissect whether the absence of a second X chromosome or the presence of a Y chromosome conferred a disadvantage on male mice, we varied sex chromosome dosage. With or without a Y chromosome, hAPP mice with one X chromosome showed worse mortality and deficits than did those with two X chromosomes. Thus, adding a second X chromosome conferred resilience to XY males and XO females. In addition, the Y chromosome, its sex-determining region Y gene (Sry), or testicular development modified mortality in hAPP mice with one X chromosome such that XY males with testicles survived longer than did XY or XO females with ovaries. Furthermore, a second X chromosome conferred resilience potentially through the candidate gene Kdm6a, which does not undergo X-linked inactivation. In humans, genetic variation in KDM6A was linked to higher brain expression and associated with less cognitive decline in aging and preclinical AD, suggesting its relevance to human brain health. Our study suggests a potential role for sex chromosomes in modulating disease vulnerability related to AD.

Tau Reduction Prevents Key Features of Autism in Mouse Models.

Autism is characterized by repetitive behaviors, impaired social interactions, and communication deficits. It is a prevalent neurodevelopmental disorder, and available treatments offer little benefit. Here, we show that genetically reducing the protein tau prevents behavioral signs of autism in two mouse models simulating distinct causes of this condition. Similar to a proportion of people with autism, both models have epilepsy, abnormally enlarged brains, and overactivation of the phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B)/ mammalian target of rapamycin (mTOR) signaling pathway. All of these abnormalities were prevented or markedly diminished by partial or complete genetic removal of tau. We identify disinhibition of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a negative PI3K regulator that tau controls, as a plausible mechanism and demonstrate that tau interacts with PTEN via tau's proline-rich domain. Our findings suggest an enabling role of tau in the pathogenesis of autism and identify tau reduction as a potential therapeutic strategy for some of the disorders that cause this condition.

GluN2A NMDA Receptor Enhancement Improves Brain Oscillations, Synchrony, and Cognitive Functions in Dravet Syndrome and Alzheimer's Disease Models.

NMDA receptors (NMDARs) play subunit-specific roles in synaptic function and are implicated in neuropsychiatric and neurodegenerative disorders. However, the in vivo consequences and therapeutic potential of pharmacologically enhancing NMDAR function via allosteric modulation are largely unknown. We examine the in vivo effects of GNE-0723, a positive allosteric modulator of GluN2A-subunit-containing NMDARs, on brain network and cognitive functions in mouse models of Dravet syndrome (DS) and Alzheimer's disease (AD). GNE-0723 use dependently potentiates synaptic NMDA receptor currents and reduces brain oscillation power with a predominant effect on low-frequency (12-20 Hz) oscillations. Interestingly, DS and AD mouse models display aberrant low-frequency oscillatory power that is tightly correlated with network hypersynchrony. GNE-0723 treatment reduces aberrant low-frequency oscillations and epileptiform discharges and improves cognitive functions in DS and AD mouse models. GluN2A-subunit-containing NMDAR enhancers may have therapeutic benefits in brain disorders with network hypersynchrony and cognitive impairments.

Klotho controls the brain-immune system interface in the choroid plexus.

Located within the brain's ventricles, the choroid plexus produces cerebrospinal fluid and forms an important barrier between the central nervous system and the blood. For unknown reasons, the choroid plexus produces high levels of the protein klotho. Here, we show that these levels naturally decline with aging. Depleting klotho selectively from the choroid plexus via targeted viral vector-induced knockout in Klothoflox/flox mice increased the expression of multiple proinflammatory factors and triggered macrophage infiltration of this structure in young mice, simulating changes in unmanipulated old mice. Wild-type mice infected with the same Cre recombinase-expressing virus did not show such alterations. Experimental depletion of klotho from the choroid plexus enhanced microglial activation in the hippocampus after peripheral injection of mice with lipopolysaccharide. In primary cultures, klotho suppressed thioredoxin-interacting protein-dependent activation of the NLRP3 inflammasome in macrophages by enhancing fibroblast growth factor 23 signaling. We conclude that klotho functions as a gatekeeper at the interface between the brain and immune system in the choroid plexus. Klotho depletion in aging or disease may weaken this barrier and promote immune-mediated neuropathogenesis.

Neuronal levels and sequence of tau modulate the power of brain rhythms.

Neural network dysfunction may contribute to functional decline and disease progression in neurodegenerative disorders. Diverse lines of evidence suggest that neuronal accumulation of tau promotes network dysfunction and cognitive decline. The A152T-variant of human tau (hTau-A152T) increases the risk of Alzheimer's disease (AD) and several other tauopathies. When overexpressed in neurons of transgenic mice, it causes age-dependent neuronal loss and cognitive decline, as well as non-convulsive epileptic activity, which is also seen in patients with AD. Using intracranial EEG recordings with electrodes implanted over the parietal cortex, we demonstrate that hTau-A152T increases the power of brain oscillations in the 0.5-6 Hz range more than wildtype human tau in transgenic lines with comparable levels of human tau protein in brain, and that genetic ablation of endogenous tau in Mapt-/- mice decreases the power of these oscillations as compared to wildtype controls. Suppression of hTau-A152T production in doxycycline-regulatable transgenic mice reversed their abnormal network activity. Treatment of hTau-A152T mice with the antiepileptic drug levetiracetam also rapidly and persistently reversed their brain dysrhythmia and network hypersynchrony. These findings suggest that both the level and the sequence of tau modulate the power of specific brain oscillations. The potential of EEG spectral changes as a biomarker deserves to be explored in clinical trials of tau-lowering therapeutics. Our results also suggest that levetiracetam treatment is able to counteract tau-dependent neural network dysfunction. Tau reduction and levetiracetam treatment may be of benefit in AD and other conditions associated with brain dysrhythmias and network hypersynchrony.

Istradefylline reduces memory deficits in aging mice with amyloid pathology.

Adenosine A2A receptors are putative therapeutic targets for neurological disorders. The adenosine A2A receptor antagonist istradefylline is approved in Japan for Parkinson's disease and is being tested in clinical trials for this condition elsewhere. A2A receptors on neurons and astrocytes may contribute to Alzheimer's disease (AD) by impairing memory. However, it is not known whether istradefylline enhances cognitive function in aging animals with AD-like amyloid plaque pathology. Here, we show that elevated levels of Aß, C-terminal fragments of the amyloid precursor protein (APP), or amyloid plaques, but not overexpression of APP per se, increase astrocytic A2A receptor levels in the hippocampus and neocortex of aging mice. Moreover, in amyloid plaque-bearing mice, low-dose istradefylline treatment enhanced spatial memory and habituation, supporting the conclusion that, within a well-defined dose range, A2A receptor blockers might help counteract memory problems in patients with Alzheimer's disease.

Expression of A152T human tau causes age-dependent neuronal dysfunction and loss in transgenic mice.

A152T-variant human tau (hTau-A152T) increases risk for tauopathies, including Alzheimer's disease. Comparing mice with regulatable expression of hTau-A152T or wild-type hTau (hTau-WT), we find age-dependent neuronal loss, cognitive impairments, and spontaneous nonconvulsive epileptiform activity primarily in hTau-A152T mice. However, overexpression of either hTau species enhances neuronal responses to electrical stimulation of synaptic inputs and to an epileptogenic chemical. hTau-A152T mice have higher hTau protein/mRNA ratios in brain, suggesting that A152T increases production or decreases clearance of hTau protein. Despite their functional abnormalities, aging hTau-A152T mice show no evidence for accumulation of insoluble tau aggregates, suggesting that their dysfunctions are caused by soluble tau. In human amyloid precursor protein (hAPP) transgenic mice, co-expression of hTau-A152T enhances risk of early death and epileptic activity, suggesting copathogenic interactions between hTau-A152T and amyloid-ß peptides or other hAPP metabolites. Thus, the A152T substitution may augment risk for neurodegenerative diseases by increasing hTau protein levels, promoting network hyperexcitability, and synergizing with the adverse effects of other pathogenic factors.

Tau reduction prevents Aß-induced axonal transport deficits by blocking activation of GSK3ß.

Axonal transport deficits in Alzheimer's disease (AD) are attributed to amyloid ß (Aß) peptides and pathological forms of the microtubule-associated protein tau. Genetic ablation of tau prevents neuronal overexcitation and axonal transport deficits caused by recombinant Aß oligomers. Relevance of these findings to naturally secreted Aß and mechanisms underlying tau's enabling effect are unknown. Here we demonstrate deficits in anterograde axonal transport of mitochondria in primary neurons from transgenic mice expressing familial AD-linked forms of human amyloid precursor protein. We show that these deficits depend on Aß1-42 production and are prevented by tau reduction. The copathogenic effect of tau did not depend on its microtubule binding, interactions with Fyn, or potential role in neuronal development. Inhibition of neuronal activity, N-methyl-d-aspartate receptor function, or glycogen synthase kinase 3ß (GSK3ß) activity or expression also abolished Aß-induced transport deficits. Tau ablation prevented Aß-induced GSK3ß activation. Thus, tau allows Aß oligomers to inhibit axonal transport through activation of GSK3ß, possibly by facilitating aberrant neuronal activity.

Life extension factor klotho prevents mortality and enhances cognition in hAPP transgenic mice.

Aging is the principal demographic risk factor for Alzheimer disease (AD), the most common neurodegenerative disorder. Klotho is a key modulator of the aging process and, when overexpressed, extends mammalian lifespan, increases synaptic plasticity, and enhances cognition. Whether klotho can counteract deficits related to neurodegenerative diseases, such as AD, is unknown. Here we show that elevating klotho expression decreases premature mortality and network dysfunction in human amyloid precursor protein (hAPP) transgenic mice, which simulate key aspects of AD. Increasing klotho levels prevented depletion of NMDA receptor (NMDAR) subunits in the hippocampus and enhanced spatial learning and memory in hAPP mice. Klotho elevation in hAPP mice increased the abundance of the GluN2B subunit of NMDAR in postsynaptic densities and NMDAR-dependent long-term potentiation, which is critical for learning and memory. Thus, increasing wild-type klotho levels or activities improves synaptic and cognitive functions, and may be of therapeutic benefit in AD and other cognitive disorders.

Astrocytic adenosine receptor A2A and Gs-coupled signaling regulate memory.

Astrocytes express a variety of G protein-coupled receptors and might influence cognitive functions, such as learning and memory. However, the roles of astrocytic Gs-coupled receptors in cognitive function are not known. We found that humans with Alzheimer's disease (AD) had increased levels of the Gs-coupled adenosine receptor A2A in astrocytes. Conditional genetic removal of these receptors enhanced long-term memory in young and aging mice and increased the levels of Arc (also known as Arg3.1), an immediate-early gene that is required for long-term memory. Chemogenetic activation of astrocytic Gs-coupled signaling reduced long-term memory in mice without affecting learning. Like humans with AD, aging mice expressing human amyloid precursor protein (hAPP) showed increased levels of astrocytic A2A receptors. Conditional genetic removal of these receptors enhanced memory in aging hAPP mice. Together, these findings establish a regulatory role for astrocytic Gs-coupled receptors in memory and suggest that AD-linked increases in astrocytic A2A receptor levels contribute to memory loss.

Network dysfunction in α-synuclein transgenic mice and human Lewy body dementia.

OBJECTIVE: Dementia with Lewy bodies (DLB) is associated with the accumulation of wild-type human α-synuclein (SYN) in neurons and with prominent slowing of brain oscillations on electroencephalography (EEG). However, it remains uncertain whether the EEG abnormalities are actually caused by SYN. METHODS: To determine whether SYN can cause neural network abnormalities, we performed EEG recordings and analyzed the expression of neuronal activity-dependent gene products in SYN transgenic mice. We also carried out comparative analyses in humans with DLB. RESULTS: We demonstrate that neuronal expression of SYN in transgenic mice causes a left shift in spectral power that closely resembles the EEG slowing observed in DLB patients. Surprisingly, SYN mice also had seizures and showed molecular hippocampal alterations indicative of aberrant network excitability, including calbindin depletion in the dentate gyrus. In postmortem brain tissues from DLB patients, we found reduced levels of calbindin mRNA in the dentate gyrus. Furthermore, nearly one quarter of DLB patients showed myoclonus, a clinical sign of aberrant network excitability that was associated with an earlier age of onset of cognitive impairments. In SYN mice, partial suppression of epileptiform activity did not alter their shift in spectral power. Furthermore, epileptiform activity in human amyloid precursor protein transgenic mice was not associated with a left shift in spectral power. INTERPRETATION: We conclude that neuronal accumulation of SYN slows brain oscillations and, in parallel, causes aberrant network excitability that can escalate into seizure activity. The potential role of aberrant network excitability in DLB merits further investigation.

Life extension factor klotho enhances cognition.

Aging is the primary risk factor for cognitive decline, an emerging health threat to aging societies worldwide. Whether anti-aging factors such as klotho can counteract cognitive decline is unknown. We show that a lifespan-extending variant of the human KLOTHO gene, KL-VS, is associated with enhanced cognition in heterozygous carriers. Because this allele increased klotho levels in serum, we analyzed transgenic mice with systemic overexpression of klotho. They performed better than controls in multiple tests of learning and memory. Elevating klotho in mice also enhanced long-term potentiation, a form of synaptic plasticity, and enriched synaptic GluN2B, an N-methyl-D-aspartate receptor (NMDAR) subunit with key functions in learning and memory. Blockade of GluN2B abolished klotho-mediated effects. Surprisingly, klotho effects were evident also in young mice and did not correlate with age in humans, suggesting independence from the aging process. Augmenting klotho or its effects may enhance cognition and counteract cognitive deficits at different life stages.

Tau reduction diminishes spatial learning and memory deficits after mild repetitive traumatic brain injury in mice.

OBJECTIVE: Because reduction of the microtubule-associated protein Tau has beneficial effects in mouse models of Alzheimer's disease and epilepsy, we wanted to determine whether this strategy can also improve the outcome of mild traumatic brain injury (TBI). METHODS: We adapted a mild frontal impact model of TBI for wildtype C57Bl/6J mice and characterized the behavioral deficits it causes in these animals. The Barnes maze, Y maze, contextual and cued fear conditioning, elevated plus maze, open field, balance beam, and forced swim test were used to assess different behavioral functions. Magnetic resonance imaging (MRI, 7 Tesla) and histological analysis of brain sections were used to look for neuropathological alterations. We also compared the functional effects of this TBI model and of controlled cortical impact in mice with two, one or no Tau alleles. RESULTS: Repeated (2-hit), but not single (1-hit), mild frontal impact impaired spatial learning and memory in wildtype mice as determined by testing of mice in the Barnes maze one month after the injury. Locomotor activity, anxiety, depression and fear related behaviors did not differ between injured and sham-injured mice. MRI imaging did not reveal focal injury or mass lesions shortly after the injury. Complete ablation or partial reduction of tau prevented deficits in spatial learning and memory after repeated mild frontal impact. Complete tau ablation also showed a trend towards protection after a single controlled cortical impact. Complete or partial reduction of tau also reduced the level of axonopathy in the corpus callosum after repeated mild frontal impact. INTERPRETATION: Tau promotes or enables the development of learning and memory deficits and of axonopathy after mild TBI, and tau reduction counteracts these adverse effects.

Human P301L-mutant tau expression in mouse entorhinal-hippocampal network causes tau aggregation and presynaptic pathology but no cognitive deficits.

Accumulation of hyperphosphorylated tau in the entorhinal cortex (EC) is one of the earliest pathological hallmarks in patients with Alzheimer's disease (AD). It can occur before significant Aß deposition and appears to "spread" into anatomically connected brain regions. To determine whether this early-stage pathology is sufficient to cause disease progression and cognitive decline in experimental models, we overexpressed mutant human tau (hTauP301L) predominantly in layer II/III neurons of the mouse EC. Cognitive functions remained normal in mice at 4, 8, 12 and 16 months of age, despite early and extensive tau accumulation in the EC. Perforant path (PP) axon terminals within the dentate gyrus (DG) contained abnormal conformations of tau even in young EC-hTau mice, and phosphorylated tau increased with age in both the EC and PP. In old mice, ultrastructural alterations in presynaptic terminals were observed at PP-to-granule cell synapses. Phosphorylated tau was more abundant in presynaptic than postsynaptic elements. Human and pathological tau was also detected within hippocampal neurons of this mouse model. Thus, hTauP301L accumulation predominantly in the EC and related presynaptic pathology in hippocampal circuits was not sufficient to cause robust cognitive deficits within the age range analyzed here.