. Gamma frequency entrainment attenuates amyloid load and modifies microglia. Nature. 2016 Dec 7;540(7632):230-235. PubMed.

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  1. This study is an ingenious attempt to determine whether interventions that stimulate fast neuronal oscillations in the brain can ameliorate Alzheimer’s pathology. The study contains four key findings:

    • 40 Hz optogenetic stimulation of parvalbumin-positive fast-spiking interneurons, which elicits gamma-frequency rhythmicity, reduced Aβ levels in pre-plaque APP mice.
    • The evoked gamma rhythm was found to enhance Aβ uptake by microglia, implying a causative relationship.
    • Visual stimulation at 40 Hz, which also elicits gamma rhythmicity, reduced endogenous mouse Aβ in wild-type mice and lowered amyloid burden in plaque-bearing APP mice.
    • The 40 Hz visual stimulation also reduced tau phosphorylation in a tau model. 

    This is one of the strongest cases made so far for the importance of neuronal network activity in modulating Aβ and tau levels, not only in the diseased brain but perhaps also under physiological conditions (see experiment in wild-type animals). One potential limitation of the study is that the gamma rhythms elicited by optogenetic stimulation or light stimulation may differ from those observed during normal behavior. Nevertheless, the study elegantly extends previous pioneering work demonstrating that neuronal activity is strongly impaired in Alzheimer’s, and that the degree of impairment correlates with the amount of amyloid and tau deposition in the brain (reviewed by Busche and Konnerth, 2016). For example, enhanced action potential firing of excitatory neurons massively increased plaque burden in APP mice (Yamamoto et al., 2015), and reducing neuronal firing rates lowered the amyloid burden (Yuan and Grutzendler, 2016). ­

    The link between increased gamma rhythmicity, microglia activation, and reduced Aβ is intriguing. I wonder what the mechanism is, since microglia are not excitable cells. A plausible hypothesis might be that a gamma-related enhanced level of overall synaptic inhibition, which likely reduces tonic hyperactivity (Busche et al., 2015) and epileptiform activity (Verret et al., 2012) in the brain, results in a reduced release of Aβ from neurons (Cirrito et al., 2005). I wonder whether the evoked gamma rhythm may also improve vascular or glymphatic clearance mechanisms.

    This new study implicates a novel treatment approach for AD that is based on the fundamental properties of brain rhythms. Such an alternative approach to conventional drug-based interventions has the advantage that it can remain non-invasive (for example, in the form of biofeedback). However, it is important to keep in mind that increased gamma rhythmicity might transmit information with heightened salience. For example, sensory signals that are normally ignored may instead be misinterpreted by the brain, resulting in misperceptions (e.g., hallucinations). Moreover, the APP mice used in this study were young; thus, a key question is whether in more advanced pathological states, when interneuron networks are more severely impaired (Verret et al., 2012), interventions that stimulate interneurons to generate gamma rhythmicity are still possible.

    References:

    . Impairments of neural circuit function in Alzheimer's disease. Philos Trans R Soc Lond B Biol Sci. 2016 Aug 5;371(1700) PubMed.

    . Chronic optogenetic activation augments aβ pathology in a mouse model of Alzheimer disease. Cell Rep. 2015 May 12;11(6):859-65. Epub 2015 Apr 30 PubMed.

    . Attenuation of β-Amyloid Deposition and Neurotoxicity by Chemogenetic Modulation of Neural Activity. J Neurosci. 2016 Jan 13;36(2):632-41. PubMed.

    . Decreased amyloid-β and increased neuronal hyperactivity by immunotherapy in Alzheimer's models. Nat Neurosci. 2015 Dec;18(12):1725-7. Epub 2015 Nov 9 PubMed.

    . Inhibitory interneuron deficit links altered network activity and cognitive dysfunction in Alzheimer model. Cell. 2012 Apr 27;149(3):708-21. PubMed.

    . Synaptic activity regulates interstitial fluid amyloid-beta levels in vivo. Neuron. 2005 Dec 22;48(6):913-22. PubMed.

    View all comments by Marc Aurel Busche
  2. In this work, Iaccarino et al. find that specific manipulation of brain gamma activity rapidly over an hour lowers Aβ40 and 42 levels as well as affects microglial gene expression in such a way as to make them more phagocytic. Over time, increasing gamma activity in 5XFAD mice was able to decrease plaque load, possibly through influencing both Aβ production and microglial function.

    This an interesting set of findings. It’s been known for some time that different manipulations of synaptic activity, pre- and postsynaptically, can alter Aβ levels acutely up or down, depending on the manipulation. However, influencing specific oscillations in the brain to see if that influences Aβ levels in such a specific way postsynaptically has not been done.

    It is interesting that the effect of gamma oscillations may be occurring via influencing neuronal Aβ production as well as via microglial clearance. The results suggest new possible ways to lower Aβ levels.

    On a related topic, work on how the sleep-wake cycle influences Aβ suggests that oscillations in the delta range that are prominent during slow-wave sleep are linked with decreased Aβ in the CNS in both animals and humans. In regard to gamma oscillations, as there are good methods to assess Aβ production and clearance in the human CNS/CSF, it will be interesting to determine if increasing gamma oscillations in the human brain also causes a decrease in Aβ levels as shown here in mice.
     

    View all comments by David Holtzman
  3. In this study, Iaccarino, Singer, and colleagues identify a novel role for hippocampal and cortical slow gamma oscillatory activity in regulating amyloid and tau pathologies in Alzheimer’s disease (AD). After identifying a deficit in slow gamma coincident with sharp-wave ripples (SWRs) in the hippocampus of 5xFAD mice, the authors show that exogenously driving slow gamma activity in the hippocampus and cortex reduces amyloid and tau pathologies. This study makes a strong case for further investigation of slow gamma oscillatory activity in the Alzheimer’s field.

    Strikingly, the hippocampal network deficits shown in this study are remarkably similar to those of an ApoE4 mouse model of AD that we published earlier this year. Specifically, we found that human ApoE4 knock-in (ApoE4-KI) mice have reduced SWR abundance and reduced slow gamma power during these SWRs (Gillespie et al., 2016). Despite the extensive pathological differences between 5xFAD mice, which likely model early onset AD, and ApoE4-KI mice, which better represent late-onset AD, this study found that 5xFAD mice have identical deficits in SWR abundance and associated slow gamma power. Further study and comparison of the underlying mechanisms that cause such a similar network impairment will be critical. The corroboration of this phenotype across disparate AD models increases the likelihood that similar hippocampal network abnormalities might exist in AD patients.

    It is important to note the distinction between slow gamma coincident with SWRs in the hippocampus and general slow gamma oscillations. The slow gamma induced by optogenetic stimulation in this study lasted much longer and had much higher consistency than physiological slow gamma activity, which fluctuates constantly in the cortex and is modulated even more rapidly in the hippocampus. While the constant stimulation was clearly effective in clearing amyloid and p-tau, it is unlikely that this exogenous slow gamma could have functionally replaced the SWR-associated slow gamma to rescue the deficit they observed. It is also critical to note the difference between hippocampal slow gamma and cortical slow gamma. Each serves unique purposes in coordinating cell assemblies for specific tasks, thus we should be cautious not to pool the CA1 and visual cortex gamma results together. However, regardless of the different types of slow gamma and their brain locations, this study demonstrates clearly that exogenously driving slow gamma in either hippocampus or cortex reduces amyloid and tau pathologies, suggesting a novel approach to target these pathologies in AD.

    Additionally, the beneficial effects of stimulating PV cells further supports the hypothesis that GABAergic interneuron dysfunction and/or death are likely involved in the pathogenesis of AD. Previous work by Verret et al. revealed a deficit in cortical gamma activity caused by PV cell dysfunction (Verret et al., 2012). Similarly, our recent study demonstrated that ApoE4 expression in GABAergic interneurons was responsible for the observed SWR-associated slow gamma deficit (Gillespie et al., 2016). It would be interesting to determine the cellular mechanism behind SWR-associated slow gamma deficits in 5xFAD mice and how it might relate to amyloid and tau pathologies and learning and memory impairments.

    This intriguing study opens many doors for future research. First, does SWR-associated slow gamma contribute to an amyloid-clearing effect as the induced slow gamma activity does? Does the power of endogenous slow gamma coincident with SWRs correlate with the extent of amyloid or tau pathology or of cognitive deficits in 5xFAD mice? Does exogenously driving slow gamma interfere with normal brain network activities and functions? This is a great start to an exciting line of inquiry.

     

    References:

    . Apolipoprotein E4 Causes Age-Dependent Disruption of Slow Gamma Oscillations during Hippocampal Sharp-Wave Ripples. Neuron. 2016 May 18;90(4):740-51. Epub 2016 May 5 PubMed.

    . Inhibitory interneuron deficit links altered network activity and cognitive dysfunction in Alzheimer model. Cell. 2012 Apr 27;149(3):708-21. PubMed.

    View all comments by Yadong Huang
  4. Roughly half of patients with AD have psychotic symptoms, i.e., delusions and/or hallucinations. Do the authors think that 40 Hz entrainment in these AD with psychosis patients might perhaps reduce their likelihood of symptoms such as hallucinations? Previously, a study found that 40 Hz stimulation partly disrupts gamma-band synchronization in schizophrenia patients who have frequent auditory hallucinations (see Koenig et al., 2012).

    Also, use of antipsychotics in AD patients increases the risk of non-cancer mortality by about 60 percent (Koponen et al., 2016). Thus, it would be great if non-invasive gamma stimulation might reduce the need for antipsychotics.

    References:

    . Is gamma band EEG synchronization reduced during auditory driving in schizophrenia patients with auditory verbal hallucinations?. Schizophr Res. 2012 Nov;141(2-3):266-70. Epub 2012 Aug 12 PubMed.

    . Risk of Mortality Associated with Antipsychotic Monotherapy and Polypharmacy Among Community-Dwelling Persons with Alzheimer's Disease. J Alzheimers Dis. 2017;56(1):107-118. PubMed.

    View all comments by Charles Stromeyer
  5. We thank the commentators for taking the time to write about these findings and Alzforum for providing a discussion forum for discoveries in Alzheimer’s disease research. The commentators bring up some excellent points that we were unable to fully discuss in the paper due to length limitations; therefore we elaborate on them here. We would also like to let researchers know that detailed methods for the LED flicker set-up will be available on the Tsai Lab website shortly.

    As both Busche and Holtzman point out, our findings build on previous studies that have shown that Aβ peptide levels are elevated following increases in neural activity and reduced following silencing of neural activity (Cirrito et al., 2003; Bero et al., 2011). Therefore, we used the random stimulation condition to control for overall changes in spiking activity caused by stimulation. We compared multi-unit firing rates during interleaved periods of 40 Hz and random stimulation and found no significant differences between firing rates in these conditions (Ext. Data Fig. 1o). Thus, while the random condition did not induce gamma oscillations, it did result in similar amounts of multi-unit spiking activity (Fig. 1e). Accordingly, we think reduced Aβ levels in response to 40 Hz stimulation were not due to decreased spiking activity.

    Because our recordings and analysis did not distinguish between pyramidal cells and FS-PV-interneurons, we cannot exclude that pyramidal cell firing rates differed between these conditions but firing of FS-PV-interneurons or other cell types masked this change. However, random optogenetic stimulation of FS-PV-interneurons provided the same amount of direct stimulation of FS-PV-interneurons yet did not reduce amyloid. In fact, optogenetic stochastic stimulation more than tripled amyloid levels while stochastic visual flicker produced no significant change, which may indicate that some aspects of the random stimulation have neurotoxic effects. While random stimulation did not result in increased gamma power, we noticed a trend of small increases in power in a wide range of frequencies, from around 20 Hz to greater than 60 Hz (Fig. 1 e). These results point to a need to understand how patterns of spiking activity affect molecular pathways and disease pathology.

    As Jones, Gillespie, and Huang describe, the deficits we find in the 5XFAD mouse model are very similar to those they found in the ApoE4 mouse and converge with evidence of gamma deficits Verret et al. report in the hAPP mouse model (Verret et al., 2012; Gillespie et al., 2016). This converging evidence from multiple mouse models of AD, including transgenic and knock-in models, suggests that these results are not due solely to overexpression of transgenes or to other side effects particular to one model. Importantly, studies in humans with AD have found altered gamma (Stam et al., 2002). Together, these results from mice and humans suggest that multiple molecular pathways that contribute to Aβ pathology may alter gamma activity in AD.

    Further study is needed to determine whether driving gamma oscillations to reduce Aβ will be therapeutic in humans, as Holtzman points out. Broadly, a current prevailing theory of AD pathogenesis points to microglia malfunction, specifically microglia’s failure to clear out pathological molecules, as a key mechanism of disease progression (Heneka et al., 2014). Therefore, interventions that recruit microglia back to an endocytotic state, as 40 Hz stimulation does, have strong therapeutic potential.

    In addition, as Busche mentions, the younger mice used in this study likely have intact interneuron networks able to entrain to 40 Hz. It will be important to determine whether methods that drive gamma oscillations will be efficient in patients and mouse models with more severe interneuron network deficiencies. Alternately, a gamma-based therapy may need to be implemented preventatively or prior to break down of interneuron networks. We look forward to a deeper investigation of both the potential and boundaries of this network-based intervention.

    References:

    . In vivo assessment of brain interstitial fluid with microdialysis reveals plaque-associated changes in amyloid-beta metabolism and half-life. J Neurosci. 2003 Oct 1;23(26):8844-53. PubMed.

    . Neuronal activity regulates the regional vulnerability to amyloid-β deposition. Nat Neurosci. 2011 Jun;14(6):750-6. PubMed.

    . Inhibitory interneuron deficit links altered network activity and cognitive dysfunction in Alzheimer model. Cell. 2012 Apr 27;149(3):708-21. PubMed.

    . Apolipoprotein E4 Causes Age-Dependent Disruption of Slow Gamma Oscillations during Hippocampal Sharp-Wave Ripples. Neuron. 2016 May 18;90(4):740-51. Epub 2016 May 5 PubMed.

    . Generalized synchronization of MEG recordings in Alzheimer's Disease: evidence for involvement of the gamma band. J Clin Neurophysiol. 2002 Dec;19(6):562-74. PubMed.

    . Innate immune activation in neurodegenerative disease. Nat Rev Immunol. 2014 Jul;14(7):463-77. PubMed.

    View all comments by Li-Huei Tsai

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