Decades before Alzheimer’s disease symptoms appear, neurons become hyperactive, and the reason could be that astrocytes fail to keep them in check. In a bioRXiv preprint uploaded on April 26, researchers led by Bart de Strooper, UK Dementia Research Institute, London, and first author Disha Shah, KU Leuven, Belgium, report that the cingulate cortex is hyperactive in cognitively normal older adults years before they develop amyloid plaques. They found the same in young APP knock-in mice. In the mouse cingulate, astrocytes did not maintain intracellular calcium signaling, but boosting calcium levels restored astrocyte activity, normalized neuron function, and calmed the hyperactive mice. This research places astrocytes into the pathological cascade much earlier than previously suspected.

  • Healthy adults have overactive cingulate neurons years before developing plaques.
  • In the same region in young APP knock-in mice, neurons are hyperactive, astrocytes sluggish.
  • Jolting astrocytes normalized mouse connectivity and behavior.

“I’m really happy that the authors focused on the role of astrocytes in brain circuit dysfunctions, because we don’t know a lot about what causes these issues,” Brian Bacskai, Harvard Medical School, told Alzforum. “The researchers used an impressive array of techniques to show that altered calcium signaling in astrocytes leads to dysfunctional neuron circuits,” he added.

In the AD brain, a collection of regions called the default mode network (DMN), which includes the prefrontal and cingulate cortices, becomes overactive years before symptom onset (Sep 2005 news; Mar 2004 news).

Shah found much the same when she analyzed functional MRI scans from healthy adults ages 50 to 80 who had enrolled in the Flemish Prevent AD Cohort. F-PACK collected baseline cognitive assessment scores, structural and functional MRI scans, and amyloid PET scans from cognitively intact people between 2009 and 2015, and is following participants for 10 years. Shah focused on 34 people who had had two amyloid scans that were captured seven years apart on average. Ten of the 34 accumulated amyloid during that time. For comparison, she matched these “accumulators” with non-accumulators with roughly the same age, sex, years of education, and baseline mini-mental state exam scores.

F-PACK measured neural activity using blood oxygen level-dependent functional MRI. BOLD detects tiny oxygen changes generated by respiratory surges in nearby neurons (Aug 2005 news). Compared to controls at baseline, people who went on to develop amyloid plaques had heightened activity in the anterior-posterior cingulate cortex (see image below), in keeping with earlier data suggesting activity changes in this region early in disease. “This brain area may be particularly vulnerable because the default mode network spends so much time in an active state when a person isn’t focusing on a task,” Shah told Alzforum.

Calm Down, Cingulate. Baseline fMRI scans reveal a hyperactive anterior cingulate cortex in people who go on to accumulate amyloid (middle), but not in those who remain amyloid-free (left). The change in amyloid (right) correlated with baseline activity in this region (red diamonds are amyloid-positives; blue, amyloid negative). [Courtesy of Shah et al., bioRXiv, 2022.]

To find out what might cause the hyperactivity if there are no plaques around, the scientists turned to mice. Shah had seen hyperactivity in homologous brain regions in APP NL-F knock-in mice and other models of amyloidosis, also before amyloid had accumulated (Shah et al., 2018; Latif-Hernandez et al., 2017; Shah et al., 2016). Bacskai and others had shown that when plaques did emerge, they intensified calcium signaling within nearby neurons and astrocytes (Sep 2008 news; Feb 2009 news). Does calcium run amok even before plaques appear?

Shah assessed intracellular calcium signaling in mice by selectively expressing the calcium indicator, GCaMP6f, in neurons or in astrocytes in the cingulate cortices of 2-month-old APP NL-F knock-ins. She then imaged the reporter using transcranial two-photon microscopy. Compared to that in wild-type mice, calcium signaling in APP NL-F neurons within the cingulate was intense, but in APP NL-F astrocytes, it was weaker.

To test whether failing astrocytic calcium signaling might explain the neuronal hyperactivity, the scientists restored astrocyte calcium signaling. They expressed hM3Dq in the cells. This Designer Receptor Exclusively Activated by Designer Drugs, aka DREADD, releases calcium through the inositol 1,4,5-trisphosphate pathway only when the mice are given the designer drug clozapine-N-oxide (CNO). In APP NL-F/hM3Dq mice, CNO boosted astrocyte calcium responses. This calmed overzealous neuronal calcium and neuronal activity in the mouse DMN, lowering the BOLD signal.

CNO also quieted neurons artificially overstimulated with dihydrokainic acid, an inhibitor of glutamate transporter-1. Astrocytes use GLT-1 to recycle glutamate from synapses, and when it is blocked, neurotransmission ramps up. Boosting astrocytic calcium signaling in dihydrokainic acid-treat mice quieted neuron activity to baseline. To the authors, this indicated that calcium signaling within astrocytes plays a key role in regulating brain network activity.

Lastly, the authors found the CNO also calmed APP NL-F behavior. These mice tend to be hyperactive, scurrying around their cages more than do wild-types. Shah found that they are also more prone to drug-induced seizures. However, once they were given CNO and the astrocytic calcium flowed, they calmed down and had fewer seizures than did untreated mice.

Why would astrocytes struggle so early in AD, without astrocytosis or amyloid plaques to cause problems? Shah thinks soluble Aβ species are to blame. “Oligomers may bind astrocyte receptors and disturb signaling cascades within the cell,” she said. In fact, Aβ dimers may block GLT-1 to prevent glutamate uptake and overexcite neurons (Aug 2019 news). Bacskai agreed that Aβ, in some form, is the likeliest culprit, but its link to malfunctioning astrocytes and neuron activity needs to be proven. “The critical role of astrocytes in brain circuit dysfunction is an avenue that needs to be explored further,” he said.—Chelsea Weidman Burke

Comments

  1. The authors provide compelling evidence for an interaction between neuronal and astrocytic dysfunction in early stages of Alzheimer’s disease and relate them to impairments of functional connectivity in mice and humans. These findings are especially relevant as they take a more integrated view of the neuro-glial circuits impaired in Alzheimer’s disease and take into account possible interactions between astrocytes and neurons.

    At early disease stages, plaques are not detectable but soluble Aβ levels are already elevated. We have previously demonstrated that Aβ dimers and/or oligomers cause an impairment of glutamate uptake via transporter proteins, which are predominantly located on astrocytic processes (Zott and Konnerth, 2022; Zott et al., 2019). The ensuing accumulation of extracellular glutamate at synapses triggers neuronal hyperactivation.

    This work by Shah et al. once more places astrocytes at the epicenter of early neuronal hyperactivity. Remarkably, the manipulation of astrocytic activity using DREADDs directly affected neuronal activity. However, the mechanism of astrocytic dysfunction in AD as well as the signaling pathway from astrocytes to neurons remain elusive. This highlights the importance to study astrocytes at early disease stages.

    Surprisingly, the authors found that astrocytic activity is downregulated in three month-old APP NL-F mice. Previously published work found no difference in astrocytic activity before (Kuchibhotla et al., 2009) and an increase in astrocytic Ca2+-transients after plaque formation (Delekate et al., 2014; Kuchibhotla et al., 2009; Reichenbach et al., 2018). However, these experiments were performed in different models and cortical regions. In consequence, longitudinal experiments to investigate the time course and spatial distribution of astrocytic dysfunctions will be crucial in the future.

    References:

    . Metabotropic P2Y1 receptor signalling mediates astrocytic hyperactivity in vivo in an Alzheimer's disease mouse model. Nat Commun. 2014 Nov 19;5:5422. PubMed.

    . Synchronous hyperactivity and intercellular calcium waves in astrocytes in Alzheimer mice. Science. 2009 Feb 27;323(5918):1211-5. PubMed.

    . P2Y1 receptor blockade normalizes network dysfunction and cognition in an Alzheimer's disease model. J Exp Med. 2018 Jun 4;215(6):1649-1663. Epub 2018 May 3 PubMed.

    . Impairments of glutamatergic synaptic transmission in Alzheimer's disease. Semin Cell Dev Biol. 2022 Mar 22; PubMed.

    . A vicious cycle of β amyloid-dependent neuronal hyperactivation. Science. 2019 Aug 9;365(6453):559-565. PubMed.

  2. It has remained largely elusive if, when, and how astrocytes modulate the activity of neuronal networks on the micro- and mesoscale level. In previous publications, we and the Bacskai lab have demonstrated that reactive astrocytes around plaques in AD mouse models show increased calcium activity, and that this hyperactivity is to a large part mediated by purinoreceptor activation in the peri-plaque pro-inflammatory environment (Kuchibhotla et al., 2009; Delekate et al., 2014). Consequently, transgenic or pharmacological normalization of this hyperactive astroglial phenotype normalizes neuronal network activity and attenuates cognitive deficits (Reichenbach et al., 2018). This was confirmed in a recent study in awake-behaving APP/PS1 mice, which showed astroglial hyperactivity at the plaque-bearing stage under baseline conditions (Lines et al., 2022). 

    This paper extends these findings, and also generates new questions. First, using resting-state fMRI, the authors identify the cingulate cortex as a region of neuronal hyperactivity (based on increased functional connectivity) in humans and transgenic APPNL-F mice at the pre- or early plaque stage. They confirm this neuronal hyperactivity by calcium imaging of the cingulate cortex in the mouse model. Surprisingly, however, they find that this hyperactive neuronal phenotype is accompanied by astroglial calcium hypoactivity, rather than hyperactivity. Stimulating astrocytes using a Designer Receptors Exclusively Activated by Designer Drugs (DREADD)-based chemogenetic approach resulted in a normalization of neuronal activity, similar to what was shown for the reduction of astroglial hyperactivity at the plaque-bearing stage in other models (Reichenbach et al., 2018). Importantly, they also show that chemogenetic stimulation of astrocytes in wild-type mice increases neuronal network activity; however, if neuronal network activity is pharmacologically raised, the same chemogenetic astroglial intervention attenuates neuronal activity.

    This paper has several important implications. First, it confirms that brain areas of aberrant network activity identified by fMRI or other whole-brain imaging techniques display network changes at the cellular and subcellular level, and that astrocytes are centrally involved in these alterations. Second, the study confirms functional and morphological studies in humans that astrocytes are among the earliest responders to rising amyloid levels (Schöll et al., 2015). Third, it implies that astrocytes possess yet-to-be-identified homoeostatic mechanisms by which their activity can raise or attenuate neuronal synaptic activity in a context-dependent fashion.

    Importantly, it appears that stimulation of pre-plaque hypoactive astrocytes has a similar effect on neuronal networks as attenuation of plaque-stage hyperactive astrocytes, suggesting a “Goldilocks” zone of astroglial activity necessary for physiological synaptic function and perhaps normal behavior.

    On the other hand, the study raises a number of puzzling questions. First, it is now firmly established that neuroinflammation is one of the earliest steps of the pathological cascade in AD, and that astrocytes respond to, and perpetuate, this inflammation. Moreover, the abovementioned studies, in addition to models of other diseases, have shown that astrocytes typically become hyperactive under pro-inflammatory conditions. Why then would they be hypoactive at the pre-plaque stage? One possibility is that specific molecular pathways, such as ATP release and purinergic activation, only appear at the plaque-bearing stage and specifically in the peri-plaque region. It will therefore be important to measure astroglial and neuronal activity in the cingulate cortex, and ideally other regions, of older APPNL-F mice as well.

    Second, the data in the current paper were obtained in anesthetized mice, and most (if not all) anesthetics profoundly decrease astrocyte signaling (Thrane et al., 2012). Although astroglial hyperactivity persisted during anesthesia in other regions in AD models (Kuchibhotla et al., 2009; Delekate et al., 2014), it is currently unknown how sedation affects astrocytes within the cingulate cortex. Specifically, their behavior during anesthesia may differ from other cortical or subcortical regions studied previously, just as default-mode network activity differs from other networks during sleep or sedation (Heine et al., 2012). 

    Third, how can one mechanism—an increase in astrocytic calcium levels—raise or attenuate neuronal network activity in a context-dependent manner? The answer is currently unknown, but may be related to different inputs to, and molecules released from, astrocytes under these conditions. These remain to be identified in future studies employing single-cell transcriptomics, transgenic manipulation, and super-resolution imaging.

    Importantly, a better understanding of the molecular mechanisms governing the modulation of neuronal network activity by astrocytes may lead to the identification of novel symptomatic or perhaps causal treatment avenues for AD.

    References:

    . Synchronous hyperactivity and intercellular calcium waves in astrocytes in Alzheimer mice. Science. 2009 Feb 27;323(5918):1211-5. PubMed.

    . Metabotropic P2Y1 receptor signalling mediates astrocytic hyperactivity in vivo in an Alzheimer's disease mouse model. Nat Commun. 2014 Nov 19;5:5422. PubMed.

    . P2Y1 receptor blockade normalizes network dysfunction and cognition in an Alzheimer's disease model. J Exp Med. 2018 Jun 4;215(6):1649-1663. Epub 2018 May 3 PubMed.

    . Astrocyte-neuronal network interplay is disrupted in Alzheimer's disease mice. Glia. 2022 Feb;70(2):368-378. Epub 2021 Nov 2 PubMed.

    . P2Y1 receptor blockade normalizes network dysfunction and cognition in an Alzheimer's disease model. J Exp Med. 2018 Jun 4;215(6):1649-1663. Epub 2018 May 3 PubMed.

    . General anesthesia selectively disrupts astrocyte calcium signaling in the awake mouse cortex. Proc Natl Acad Sci U S A. 2012 Nov 13;109(46):18974-9. Epub 2012 Oct 29 PubMed.

    . Resting state networks and consciousness: alterations of multiple resting state network connectivity in physiological, pharmacological, and pathological consciousness States. Front Psychol. 2012;3:295. Epub 2012 Aug 27 PubMed.

  3. Our results, and a recent paper from Lee et al. (2022), report a decrease of astrocyte calcium activity at early stages of amyloid pathology. This is in contrast with observations of increased calcium signaling reported in plaque-bearing mice (Kuchibhotla et al., 2009Lines et al., 2022; Delekate et al., 2014). We argue that the decrease of astrocyte calcium signaling is a phenotype attributed to preplaque stages of the disease, recovery of which improves AD-related network disruptions. We highly recommend the preprint article at Cell Reports by Lee at al., who made similar observations independently in an APP/PS1 mouse model of amyloid pathology. That our findings are consistent across very different mouse models, and can be reproduced by independent research groups, indicates these results are robust and further emphasizes their importance.

    References:

    . Synchronous hyperactivity and intercellular calcium waves in astrocytes in Alzheimer mice. Science. 2009 Feb 27;323(5918):1211-5. PubMed.

    . Astrocyte-neuronal network interplay is disrupted in Alzheimer's disease mice. Glia. 2022 Feb;70(2):368-378. Epub 2021 Nov 2 PubMed.

    . Metabotropic P2Y1 receptor signalling mediates astrocytic hyperactivity in vivo in an Alzheimer's disease mouse model. Nat Commun. 2014 Nov 19;5:5422. PubMed.

    . Optogenetic Targeting of Astrocytes Restores Sleep-Dependent Brain Rhythm Function and Slows Alzheimer's Disease. Cell Reports, 8 Apr 2022 Cell Reports

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References

News Citations

  1. Network News: Images of AD Brains Reveal Widespread Snafus
  2. Network Diagnostics: "Default-Mode" Brain Areas Identify Early AD
  3. MRI—The Good, the Bad and the…BOLD?
  4. Hyperactive Neurons and Amyloid, Side by Side
  5. Making Waves—Calcium Dysregulation in Astrocytes of AD Mice
  6. Aβ Dimers Block Glutamate Uptake, Fire Up Synapses

Research Models Citations

  1. APP NL-F Knock-in

Paper Citations

  1. . Spatial reversal learning defect coincides with hypersynchronous telencephalic BOLD functional connectivity in APPNL-F/NL-F knock-in mice. Sci Rep. 2018 Apr 19;8(1):6264. PubMed.
  2. . Subtle behavioral changes and increased prefrontal-hippocampal network synchronicity in APPNL-G-F mice before prominent plaque deposition. Behav Brain Res. 2017 Nov 20; PubMed.
  3. . Early pathologic amyloid induces hypersynchrony of BOLD resting-state networks in transgenic mice and provides an early therapeutic window before amyloid plaque deposition. Alzheimers Dement. 2016 Sep;12(9):964-76. Epub 2016 Apr 21 PubMed.

Further Reading

Papers

  1. . Optogenetic Targeting of Astrocytes Restores Sleep-Dependent Brain Rhythm Function and Slows Alzheimer's Disease. Cell Reports, 8 Apr 2022 Cell Reports
  2. . Neuronal hyperactivity--A key defect in Alzheimer's disease?. Bioessays. 2015 Jun;37(6):624-32. Epub 2015 Mar 14 PubMed.

Primary Papers

  1. . Astrocyte calcium dysfunction causes early network hyperactivity in Alzheimer’s Disease. bioRxiv, April 26, 2022 bioRxiv