Julia R. Clarke

Inflammation, defective insulin signaling, and neuronal dysfunction in Alzheimer's disease

A link between Alzheimers disease (AD) and metabolic disorders has been established, with patients with type 2 diabetes at increased risk of developing AD and vice versa. The incidence of metabolic disorders, including insulin resistance and type 2 diabetes is increasing at alarming rates worldwide, primarily as a result of poor lifestyle habits. In parallel, as the world population ages, the prevalence of AD, the most common form of dementia in the elderly, also increases. In addition to their epidemiologic and clinical association, mounting recent evidence indicates shared mechanisms of pathogenesis between metabolic disorders and AD. We discuss the concept that peripheral and central nervous system inflammation link the pathogenesis of AD and metabolic diseases. We also explore the contribution of brain inflammation to defective insulin signaling and neuronal dysfunction. Last, we review recent evidence indicating that targeting neuroinflammation may provide novel therapeutic avenues for AD.

TNF-α mediates PKR-dependent memory impairment and brain IRS-1 inhibition induced by Alzheimer's β-amyloid oligomers in mice and monkeys

Alzheimers disease (AD) and type 2 diabetes appear to share similar pathogenic mechanisms. dsRNA-dependent protein kinase (PKR) underlies peripheral insulin resistance in metabolic disorders. PKR phosphorylates eukaryotic translation initiation factor 2α (eIF2α-P), and AD brains exhibit elevated phospho-PKR and eIF2α-P levels. Whether and how PKR and eIF2α-P participate in defective brain insulin signaling and cognitive impairment in AD are unknown. We report that β-amyloid oligomers, AD-associated toxins, activate PKR in a tumor necrosis factor α (TNF-α)-dependent manner, resulting in eIF2α-P, neuronal insulin receptor substrate (IRS-1) inhibition, synapse loss, and memory impairment. Brain phospho-PKR and eIF2α-P were elevated in AD animal models, including monkeys given intracerebroventricular oligomer infusions. Oligomers failed to trigger eIF2α-P and cognitive impairment in PKR(-/-) and TNFR1(-/-) mice. Bolstering insulin signaling rescued phospho-PKR and eIF2α-P. Results reveal pathogenic mechanisms shared by AD and diabetes and establish that proinflammatory signaling mediates oligomer-induced IRS-1 inhibition and PKR-dependent synapse and memory loss.

Alzheimer‐associated Aβ oligomers impact the central nervous system to induce peripheral metabolic deregulation

Alzheimers disease (AD) is associated with peripheral metabolic disorders. Clinical/epidemiological data indicate increased risk of diabetes in AD patients. Here, we show that intracerebroventricular infusion of AD‐associated Aβ oligomers (AβOs) in mice triggered peripheral glucose intolerance, a phenomenon further verified in two transgenic mouse models of AD. Systemically injected AβOs failed to induce glucose intolerance, suggesting AβOs target brain regions involved in peripheral metabolic control. Accordingly, we show that AβOs affected hypothalamic neurons in culture, inducing eukaryotic translation initiation factor 2α phosphorylation (eIF2α‐P). AβOs further induced eIF2α‐P and activated pro‐inflammatory IKKβ/NF‐κB signaling in the hypothalamus of mice and macaques. AβOs failed to trigger peripheral glucose intolerance in tumor necrosis factor‐α (TNF‐α) receptor 1 knockout mice. Pharmacological inhibition of brain inflammation and endoplasmic reticulum stress prevented glucose intolerance in mice, indicating that AβOs act via a central route to affect peripheral glucose homeostasis. While the hypothalamus has been largely ignored in the AD field, our findings indicate that AβOs affect this brain region and reveal novel shared molecular mechanisms between hypothalamic dysfunction in metabolic disorders and AD.

Memantine Rescues Transient Cognitive Impairment Caused by High-Molecular-Weight Aβ Oligomers But Not the Persistent Impairment Induced by Low-Molecular-Weight Oligomers

Brain accumulation of soluble amyloid-β oligomers (AβOs) has been implicated in synapse failure and cognitive impairment in Alzheimers disease (AD). However, whether and how oligomers of different sizes induce synapse dysfunction is a matter of controversy. Here, we report that low-molecular-weight (LMW) and high-molecular-weight (HMW) Aβ oligomers differentially impact synapses and memory. A single intracerebroventricular injection of LMW AβOs (10 pmol) induced rapid and persistent cognitive impairment in mice. On the other hand, memory deficit induced by HMW AβOs (10 pmol) was found to be reversible. While memory impairment in LMW oligomer-injected mice was associated with decreased hippocampal synaptophysin and GluN2B immunoreactivities, synaptic pathology was not detected in the hippocampi of HMW oligomer-injected mice. On the other hand, HMW oligomers, but not LMW oligomers, induced oxidative stress in hippocampal neurons. Memantine rescued both neuronal oxidative stress and the transient memory impairment caused by HMW oligomers, but did not prevent the persistent cognitive deficit induced by LMW oligomers. Results establish that different Aβ oligomer assemblies act in an orchestrated manner, inducing different pathologies and leading to synapse dysfunction. Furthermore, results suggest a mechanistic explanation for the limited efficacy of memantine in preventing memory loss in AD.

Amyloid-β oligomers link depressive-like behavior and cognitive deficits in mice

Depression is one of the most common psychiatric symptoms in Alzheimers disease (AD), and considerable evidence indicates that major depressive disorder increases the risk of AD.1, 2, 3 To date, however, the molecular mechanisms underlying the clinical association between depression and AD have remained elusive. Soluble oligomers of the amyloid-β peptide (AβOs) accumulate in the brains of AD patients and are increasingly recognized as the proximal neurotoxins responsible for synapse failure and memory deficits in AD.4, 5 We have hypothesized that AβOs might be mechanistically linked to behavioral changes in AD. In order to test this hypothesis, mice were given a single intracerebroventricular (i.c.v.) injection of 10 pmol AβOs and were subsequently evaluated in the Porsolt forced swim test (FST) for assessment of depressive-like behavior. Compared with vehicle-injected control mice, AβO-injected mice exhibited a significant increase in immobility in the FST, both 24 h and 8 days after AβO injections (Figure 1a). Similar results were obtained when animals were assessed in the tail suspension test, another classical task to evaluate depressive-like behavior in rodents (Supplementary Figure S1a). AβO-induced immobility in the FST was blocked by anti-depressant (fluoxetine) treatment (Figure 1a). An important feature of depressive disorder is anhedonic behavior, including decreased interest for pleasurable sensorial experiences. Whereas vehicle-injected mice exhibited an expected preference for sucrose solution over plain water, AβO-injected mice did not exhibit such preference, indicating that AβOs instigate anhedonic behavior (Figure 1b). Figure 1 Amyloid-β oligomers (AβOs) induce depressive-like behavior, memory deficits and hippocampal recruitment of microglia and astrocytes in mice. Three-month-old Swiss mice received a single intracerebroventricular (i.c.v.) injection of AβOs ... As memory deficit is the main clinical symptom of AD, we investigated the impact of AβOs on mice memory using the novel object recognition (OR) task. Results showed that 24 h after i.c.v. injection, AβO-treated mice spent equal amounts of time exploring both old (familiar) and new (novel) objects, indicating a deficit in declarative recognition memory, whereas vehicle-injected animals exhibited a significant preference for the novel object (Figure 1c). Treatment with fluoxetine prevented the memory deficit induced by AβOs (Figure 1c). Control measurements showed no changes in spontaneous exploratory or locomotor activities of fluoxetine-, saline-, vehicle- or AβO-injected animals during the training phase of the OR test (Supplementary Figure S1c–e). As the hippocampus is a key anatomical structure for OR memory, we sought to determine whether AβOs injected i.c.v. reached the hippocampus. Indeed, robust AβO immunoreactivity was verified using an anti-oligomer monoclonal antibody (NU4)6 in hippocampi from AβO-injected mice, but not in hippocampi from control vehicle-injected animals (Supplementary Figure S1b). Together, these results indicate that AβOs have an acute impact on memory, learning and mood in mice, and that fluoxetine treatment prevented both cognitive impairment and depressive-like behavior induced by AβOs. The beneficial actions of fluoxetine have been partly ascribed to its anti-inflammatory effect. This led us to ask whether AβOs triggered an inflammatory response in the mouse brain. The brains of mice used in the tests described above were analyzed for levels of pro-inflammatory cytokines after i.c.v. injection of AβOs or vehicle. AβO-injected animals showed significantly elevated brain levels of interleukin-1β and tumor necrosis factor-α compared with vehicle-injected animals (Figure 1d, e). Sections from the hippocampus and cortex of AβO- or vehicle-injected mice were further immunostained for the presence of microglia (anti-Iba-1 antibody) and astrocytes (anti-GFAP antibody). Compared with vehicle-injected animals, AβO-injected mice showed markedly increased immunoreactivities for both Iba-1 and GFAP in the hippocampus and cortex 24 h after injection (Figure 1f–o, and Supplementary Figure 1f–o). The increase in glial cell numbers instigated by AβOs was blocked by fluoxetine treatment of the animals before AβO injection (Figure 1f–o, and Supplementary Figure 1f–o). The current findings establish that AβOs link memory impairment and depressive-like behavior in mice, providing molecular mechanistic support to clinical evidence connecting AD and depressive disorder. The impact of AβOs on mood, learning and memory, and its prevention by fluoxetine, can likely be attributed to the activation of inflammatory pathways (as shown here) and, possibly, to the deregulation of the serotonergic axis. The latter possibility is in line with the observation that pro-inflammatory cytokines impact serotonin metabolism7, 8 and that increased serotonin levels are associated with lower brain Aβ levels in transgenic mouse models of AD and in humans.9 Moreover, 5-HT1A and 5-HT2A receptors have been reported to be reduced in post-mortem AD brain,10 and 5-HT1A receptors have been found to be reduced in vivo in AD.11 Brain disturbances that place a person at risk for developing depression and AD are still largely unknown. By revealing that AβOs underlie both cognitive and depressive-like symptoms in mice, our results suggest a mechanism by which elevated brain levels of AβOs may be linked to changes in cognition and mood in AD.

Interaction of amyloid-β (Aβ) oligomers with neurexin 2α and neuroligin 1 mediates synapse damage and memory loss in mice

Brain accumulation of the amyloid-β protein (Aβ) and synapse loss are neuropathological hallmarks of Alzheimer disease (AD). Aβ oligomers (AβOs) are synaptotoxins that build up in the brains of patients and are thought to contribute to memory impairment in AD. Thus, identification of novel synaptic components that are targeted by AβOs may contribute to the elucidation of disease-relevant mechanisms. Trans-synaptic interactions between neurexins (Nrxs) and neuroligins (NLs) are essential for synapse structure, stability, and function, and reduced NL levels have been associated recently with AD. Here we investigated whether the interaction of AβOs with Nrxs or NLs mediates synapse damage and cognitive impairment in AD models. We found that AβOs interact with different isoforms of Nrx and NL, including Nrx2α and NL1. Anti-Nrx2α and anti-NL1 antibodies reduced AβO binding to hippocampal neurons and prevented AβO-induced neuronal oxidative stress and synapse loss. Anti-Nrx2α and anti-NL1 antibodies further blocked memory impairment induced by AβOs in mice. The results indicate that Nrx2α and NL1 are targets of AβOs and that prevention of this interaction reduces the deleterious impact of AβOs on synapses and cognition. Identification of Nrx2α and NL1 as synaptic components that interact with AβOs may pave the way for development of novel approaches aimed at halting synapse failure and cognitive loss in AD.

Chronic sleep restriction promotes brain inflammation and synapse loss, and potentiates memory impairment induced by amyloid-β oligomers in mice

It is increasingly recognized that sleep disturbances and Alzheimers disease (AD) share a bidirectional relationship. AD patients exhibit sleep problems and alterations in the regulation of circadian rhythms; conversely, poor quality of sleep increases the risk of development of AD. The aim of the current study was to determine whether chronic sleep restriction potentiates the brain impact of amyloid-β oligomers (AβOs), toxins that build up in AD brains and are thought to underlie synapse damage and memory impairment. We further investigated whether alterations in levels of pro-inflammatory mediators could play a role in memory impairment in sleep-restricted mice. We found that a single intracerebroventricular (i.c.v.) infusion of AβOs disturbed sleep pattern in mice. Conversely, chronically sleep-restricted mice exhibited higher brain expression of pro-inflammatory mediators, reductions in levels of pre- and post-synaptic marker proteins, and exhibited increased susceptibility to the impact of i.c.v. infusion of a sub-toxic dose of AβOs (1pmol) on performance in the novel object recognition memory task. Sleep-restricted mice further exhibited an increase in brain TNF-α levels in response to AβOs. Interestingly, memory impairment in sleep-restricted AβO-infused mice was prevented by treatment with the TNF-α neutralizing monoclonal antibody, infliximab. Results substantiate the notion of a dual relationship between sleep and AD, whereby AβOs disrupt sleep/wake patterns and chronic sleep restriction increases brain vulnerability to AβOs, and point to a key role of brain inflammation in increased susceptibility to AβOs in sleep-restricted mice.

Brain infusion of α-synuclein oligomers induces motor and non-motor Parkinson’s disease-like symptoms in mice

Abstract Parkinson’s disease (PD) is characterized by motor dysfunction, which is preceded by a number of non‐motor symptoms including olfactory deficits. Aggregation of &agr;‐synuclein (&agr;‐syn) gives rise to Lewy bodies in dopaminergic neurons and is thought to play a central role in PD pathology. However, whether amyloid fibrils or soluble oligomers of &agr;–syn are the main neurotoxic species in PD remains controversial. Here, we performed a single intracerebroventricular (i.c.v.) infusion of &agr;‐syn oligomers (&agr;‐SYOs) in mice and evaluated motor and non‐motor symptoms. Familiar bedding and vanillin essence discrimination tasks showed that &agr;‐SYOs impaired olfactory performance of mice, and decreased TH and dopamine levels in the olfactory bulb early after infusion. The olfactory deficit persisted until 45 days post‐infusion (dpi). &agr;‐ SYO‐infused mice behaved normally in the object recognition and forced swim tests, but showed increased anxiety‐like behavior in the open field and elevated plus maze tests 20 dpi. Finally, administration of &agr;‐SYOs induced late motor impairment in the pole test and rotarod paradigms, along with reduced TH and dopamine content in the caudate putamen, 45 dpi. Reduced number of TH‐positive cells was also seen in the substantia nigra of &agr;‐SYO‐injected mice compared to control. In conclusion, i.c.v. infusion of &agr;‐SYOs recapitulated some of PD‐associated non‐motor symptoms, such as increased anxiety and olfactory dysfunction, but failed to recapitulate memory impairment and depressive‐like behavior typical of the disease. Moreover, &agr;‐SYOs i.c.v. administration induced motor deficits and loss of TH and dopamine levels, key features of PD. Results point to &agr;‐syn oligomers as the proximal neurotoxins responsible for early non‐motor and motor deficits in PD and suggest that the i.c.v. infusion model characterized here may comprise a useful tool for identification of PD novel therapeutic targets and drug screening.

The diabetes drug liraglutide reverses cognitive impairment in mice and attenuates insulin receptor and synaptic pathology in a non-human primate model of Alzheimer's disease: Liraglutide protects memory, insulin receptors, and synapses

Alzheimers disease (AD) is a devastating neurological disorder that still lacks an effective treatment, and this has stimulated an intense pursuit of disease‐modifying therapeutics. Given the increasingly recognized link between AD and defective brain insulin signaling, we investigated the actions of liraglutide, a glucagon‐like peptide‐1 (GLP‐1) analog marketed for treatment of type 2 diabetes, in experimental models of AD. Insulin receptor pathology is an important feature of AD brains that impairs the neuroprotective actions of central insulin signaling. Here, we show that liraglutide prevented the loss of brain insulin receptors and synapses, and reversed memory impairment induced by AD‐linked amyloid‐β oligomers (AβOs) in mice. Using hippocampal neuronal cultures, we determined that the mechanism of neuroprotection by liraglutide involves activation of the PKA signaling pathway. Infusion of AβOs into the lateral cerebral ventricle of non‐human primates (NHPs) led to marked loss of insulin receptors and synapses in brain regions related to memory. Systemic treatment of NHPs with liraglutide provided partial protection, decreasing AD‐related insulin receptor, synaptic, and tau pathology in specific brain regions. Synapse damage and elimination are amongst the earliest known pathological changes and the best correlates of memory impairment in AD. The results illuminate mechanisms of neuroprotection by liraglutide, and indicate that GLP‐1 receptor activation may be harnessed to protect brain insulin receptors and synapses in AD.

Acute and chronic neurological consequences of early-life Zika virus infection in mice

Infection by Zika virus triggers epileptic seizures in newborn mice and leads to viral persistence in the brain and behavioral deficits in adulthood. Zika leaves lasting impact on brain Perinatal Zika virus (ZIKV) infection has been associated with brain alterations in newborns. However, whether ZIKV exposure during development has long-term neurological consequences is not completely understood. Nem de Oliveira Souza et al. report that newborn mice infected with ZIKV develop acute brain abnormalities. During adulthood, perinatally infected mice showed persistent viral replication, neuropathological alterations, behavioral impairments, and altered brain excitability. Blocking tumor necrosis factor–α (TNF-α) early after infection prevented this hyperexcitability in mouse brain. The results suggest that anti-inflammatory treatments might be used to prevent the persistent increase in neuronal excitability induced by ZIKV infection in brain tissue. Although congenital Zika virus (ZIKV) exposure has been associated with microcephaly and other neurodevelopmental disorders, long-term consequences of perinatal infection are largely unknown. We evaluated short- and long-term neuropathological and behavioral consequences of neonatal ZIKV infection in mice. ZIKV showed brain tropism, causing postnatal-onset microcephaly and several behavioral deficits in adulthood. During the acute phase of infection, mice developed frequent seizures, which were reduced by tumor necrosis factor–α (TNF-α) inhibition. During adulthood, ZIKV replication persisted in neonatally infected mice, and the animals showed increased susceptibility to chemically induced seizures, neurodegeneration, and brain calcifications. Altogether, the results show that neonatal ZIKV infection has long-term neuropathological and behavioral complications in mice and suggest that early inhibition of TNF-α–mediated neuroinflammation might be an effective therapeutic strategy to prevent the development of chronic neurological abnormalities.