In vivo modulation of hippocampal excitability by M4 muscarinic

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In vivo modulation of hippocampal excitability by M4 muscarinic acetylcholine receptor activator: implications for treatment of Alzheimer's disease and schizophrenic patients Michael Popiolek, Yael Mandelblat-Cerf, Damon Young, Jonathan Garst-Orozco, Susan M Lotarski, Eda Stark, Melissa Kramer, Christopher R. Butler, and Rouba Kozak ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00496 • Publication Date (Web): 17 Oct 2018 Downloaded from http://pubs.acs.org on October 20, 2018

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ACS Chemical Neuroscience

Title: In vivo modulation of hippocampal excitability by M4 muscarinic acetylcholine receptor activator: implications for treatment of Alzheimer's disease and schizophrenic patients Authors: Michael Popiolek1,4*, Yael Mandelblat-Cerf1,5, Damon Young1,5, Jonathan GarstOrozco1, Susan M. Lotarski1,4, Eda Stark1, Melissa Kramer2, Christopher R. Butler3, Rouba Kozak1,5 1Internal

Medicine Research Unit, 2Pharmacokinetics, Dynamics and Metabolism, 3Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development, Cambridge, Massachusetts 02139. 4Current address: Sage Therapeutics, 215 First St Suite 220, Cambridge, MA 02142 5Current address: Biogen, Cambridge, MA 02142 *corresponding author: [email protected]

Funding source statement: At the time that this work was performed all authors were employees of Pfizer Inc. Authors do not declare any conflict of interest. PT-3763 was synthesized by Pfizer. Abbreviations: BQL, below quantifiable level; CA1, Cornu Ammonis 1; CA3, Cornu Ammonis 3; DG, dente gyrus; fEPSP, field excitatory postsynaptic potential; mAChR, muscarinic acetylcholine receptor; MCI, mild cognitive impairment; MWM, Morris water maze; PAM, positive allosteric modulator; sc, subcutaneous Author contributions: MP, YMC and RK wrote the article; YMC, DY, JGO, SML, ES and MK run studies supporting this article; CB designed PT-3763; MP and RK designed the research; MP, YMC, DY, JGO, SML and ES analyzed the data

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Abstract: Abnormal hippocampal activity has been linked to impaired cognitive performance in Alzheimer’s disease and schizophrenia leading to a hypothesis that normalization of this activity may be therapeutically beneficial. Our work sugests that one approach for hippocampal normalization maybe through activation of the M4 muscarinic acetylcholine receptor. We used a brain penetrant M4 muscarinic acetylcholine receptor selective activator, PT-3763, to show dosedependent attenuation of field potentials in Schaffer collateral (CA3-CA1) and recurrent associational connections (CA3-CA3) ex vivo in hippocampal slices. In vivo, systemic administration of PT-3763 led to attenuation of glutamate release in CA3 as measured by amperometry, and to a dose-dependent decrease in population CA1 pyramidal activity as measured by fiber photometry. This decrease in population activity was also evident with a localized administration of the compound to the recorded site. Finally, PT-3763 reversed scopolamine-induced deficit in Morris water maze. Our results suggest that M4 muscarinic acetylcholine receptor activation may be a suitable therapeutic treatment in diseases associated with hyperactive hippocampal activity. Keywords: M4 muscarinic acetylcholine receptor, Alzheimer’s disease, schizophrenia, MCI, hippocampal hyperactivity, hippocampus For table of contents use only:


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Introduction: Alzheimer’s disease and schizophrenia are two devastating brain disorders with substantial unmet medical need.

For Alzheimer’s, disease-modifying treatment, especially focused on

amyloid- clearance, became the centerpiece of therapeutic approach. Unfortunately, despite lowering amyloid- load in mild to moderate Alzheimer disease patients, every treatment thus far failed primary clinical endpoint1. Most of the approved treatments for schizophrenia, at least in part, rely on targeting the dopamine 2 receptors. While these antipsychotic drugs show some efficacy on positive symptoms other domains including negative and cognitive impairments are not satisfactorily treated2. Preclinical and clinical evidence suggests that neuronal circuits become hyperactive in the early stages of the Alzheimer’s disease contributing to disease progression

3,4.

Hippocampal

hyperactivity is most pronounced in mild cognitive impairment (MCI) patients and correlates with poor cognitive performance 5. Furthermore, few studies suggest that neuronal hyperactivity may increase the release and pathology of tau

6

and amyloid-

7,8.

In fact, number of studies

demonstrated normalization of the hippocampal hyperactivity in MCI patients lead to improved



performance on cognitive tasks 9, although it is not yet know if such treatment will delay disease onset. Atrophy of the cholinergic system has been well documented in Alzheimer’s disease 10, which in part, can be explained by sensitivity of the cholinergic synapses to the neurotoxic effect of amyloid- 11. While the cholinergic system undergoes neurodegeneration limiting cholinergic tone, the neurons expressing muscarinic acetylcholine receptors (mAChRs) including the M4 mAChR, have been found to be largely intact in early and mid-stages Alzheimer’s disease 12. A strategy focused on preserving activity of the mAChRs through positive allosteric modulators (PAMs) may alleviate loss of cholinergic tone as it enables activation of these receptors despite diminishing cholinergic tone. Restored M4 mAChR signaling, in turn, may restore balanced hippocampal activity, leading to improved functional connectivity and delayed disease onset. Multiple brain regions have been implicated in pathophysiology of schizophrenia with abnormal functional connectivity between cortical and subcortical regions 13. Because of the connectivity, intervation in one region may propage and restore balance in the network. One such region is the hippocampous where hyperactivity was hypothesized to contribute to psychosis and cognitive impairment in schizophrenia based on transcriptional, morphological and functional changes observed in schizophrenic brains 14,15. The NR1 mRNA was found to be significantly decreased within the dente gyrus (DG) 16,17, while NR2B mRNA expression was significantly increased within the CA3 of the schizophrenic patients 16,18 suggesting that hypoactivity within DG results in hyperactivity in CA3. Supporting this hypothesis, decreased mossy fiber terminals synapses (DGCA3), volume fraction of spines and total number of invaginated spines were observed in schizophrenic patients 19. While not consistent across all studies, many clinical reports observed statistically significant increase of hippocampal resting


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stage fMRI 20–24 in schizophrenic patients. This data led to the proposal that attenuation of hyperactive CA3/CA1 will lead to antipsychotic and procognitive efficacy 15. Activation of the M4 mAChR has been demonstrated to lead to attenuation of hippocampal activity ex vivo 25,26, which led us to hypothesize that selective activation of the M4 mAChR may be therapeutically beneficial. In our studies, we used previously described PT-3763, an M4 mAChR selective PAM (rat M4 mAChR EC50 of 0.81 nM and human M2 mAChR EC50 of 1.277 M) 26, to quantify impact on hippocampal circuitry ex vivo and in vivo. PT-3763 led to a dosedependent attenuation of Schaffer collateral (CA3CA1) and recurrent associational connection (CA3-CA3) field excitatory postsynaptic potentials (fEPSPs) ex vivo. Consistent with this ex vivo activity, PT-3763 led to a suppression of hippocampal pyramidal activity in vivo in freely moving mice as measured by fiber photometry in CA1 and an attenuation of glutamate release in CA3, seen using amperometry. Ultimately, treatment with PT-3763 led to improvement on Morris water maze, a hippocampal mediated task 27. Collectively, this data supports therapeutic potential of M4 mAChR PAM and warrants further development. Results: Hippocampal slice recordings Previously we disclosed in vitro selectivity of PT-3763 for the M4 mAChR over other muscarinic receptor subtypes 26. In this report we focused on using PT-3763 to first confirm that application of this M4 mAChR PAM can attenuate synaptic transmission in the Schaffer collateral pathway (CA3 to CA1 connectivity) 28 as has been demonstrated within hippocampal slice electrophysiology with this 26 and other M4 mAChR PAMs 25,29. Consistent with prior observations, PT-3763 led to a dose-dependent reduction of fEPSPs evoked by electrical



stimulation of axons in stratum radiatum with an IC50 of 15.63 nM (95% confidence interval of 9.01 nM to 27.16 nM) when co-administered with an EC20 of oxotremorine (20 nM), as this PAM potentiates efficacy of the orthosteric agonist (Figure 1A). We hypothesized that the reduction in transmission in the Schaffer collateral pathway is the consequence of M4 mAChR activation on the presynaptic axonal termini of CA3 pyramidal neurons. Since CA3 axons synapse onto CA3, connection referred to as the recurrent associational connections 30, we stimulated and recorded hippocampal fEPSPs within CA3. As expected, PT-3763 led to a dose-dependent reduction of CA3 field excitability with an IC50 of 7.09 nM (95% confidence interval of 1.83 nM to 27.35 nM) (Figure 1B). The potency of the Schaffer collateral and the association connections were very comparable reinforcing that PT3763 mediated its effect through activation of the M4 mAChR in both readouts. Figure 1: Hippocampal slice electrophysiology. A) A dose-dependent suppression of the Schaffer collateral (CA3) input to CA1 synaptic transmission was observed through M4 mAChR activation with PT-3763 (n =5) as measured by fEPSP. To verify that the suppression recorded in CA1 is mediated by activity in CA3, stimulation and recording within CA3 was performed. B) A dose-dependent suppression was observed on CA3 after application of PT-3763 (n = 5). Both readouts were performed in presence of EC20 of oxotremorine (20 nM) A

B CA3-CA1 Slice fEPSP

CA3-CA3 Slice fEPSP 1.2

Normalized Response

1.2

Normalized Response

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0.8

0.4

IC50 0.0 -10

-9

-8

1.563e-008 -7

Log [PT-3763], M

-6

-5

0.8

0.4

IC50 0.0 -10

-9

-8

7.091e-009 -7

Log [PT-3763], M


-6

-5

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Pharmacokinetics Having established hippocampal ex vivo activity with PT-3763 we wanted to assess if we could observe hippocampal effect with PT-3763 in vivo on mouse fiber photometry, rat amperometry and rat Morris water maze. To that end, brain penetrance was assessed by quantifying unbound brain exposure of PT-3763 after subcutaneous administration (sc). A dosedependent exposure was observed in both mouse and rat when PT-3763 was dosed from 0.32 to 32 mg/kg in half-log dilutions (Table 1). Table 1: PT-3763 is brain penetrant. Brain exposure of PT-3763 in mouse and rat was quantified 30 minutes post subcutaneous administration (n = 3 per species). BQL = below quantifiable level; extrapolated data demarcated by # (data extrapolated from a collection done 60 min post systemic drug administration where the 0.32 mg/kg dose led to 2.2 nM unbound brain exposure and 1.0 mg/kg dose led to 6.5 nM unbound brain exposure). Unbound brain exposure (nM) Dose

Mouse @ 30 min Rat @ 30 min

0.32 mg/kg

BQL

2.8#

1.0 mg/kg

BQL

8.2#

3.2 mg/kg

22.6

15.0

10 mg/kg

59.6

30.6

32 mg/kg

94.0

84.7

Fiber photometry We tested if application of PT-3763 results in a decreased activity of CA1 pyramidal neurons in vivo as it has ex vivo. Using fiber photometry, we monitored the aggregate calcium activity of populations of pyramidal neurons in eight freely moving mice. Experiments were conducted in an open arena, and mice were habituated to the recording paradigm (see Methods).



Photometry signal was continuously recorded from 10 minutes prior to injection to 30 minutes post injection. The recordings revealed a significant dose response decrease in population CA1 pyramidal activity following injection of M4 PAM (Figure 2A and 2B), as tested by the activity 5 minutes post injection (p=0.009, Multiway Anova). The dose response effect lasted 30 minutes post injection (p=0.004, Multiway Anova). To directly test the effect of M4 PAM on hippocampal neuronal activity, a subset of mice (n=3) were implanted with a cannula attached to the optic fiber. Drug administration directly to the recorded area resulted in a sharp drop in activity (Figure 2C and 2D). We were concerned that the drop-in activity may merely be a result of the volume injected to the area that could obscure light transition or stress the neurons. However, administration of aCSF in the same volume (0.5 l) resulted in a minimal drop relative to the sharp drop after M4 PAM (Figure 2C and 2D, blue as compare to red, p=0.00191) suggesting that the effect was related to treatment with the M4 PAM. Figure 2: Activation of M4 mAChR through application of PT-3763 suppresses hippocampal activity in vivo in freely moving mice as measured by calcium fluorescence indicator (GCaMP6f) with fiber photometry. A) A dose-dependent suppression of population activity was observed after systemic administration of PT-3763 in CA1 (n=8). B) Representative traces after systemic administration of PT-3763. C) To assess if this effect is mediated by hippocampal network or only influenced by other brain regions projecting into the hippocampus, a localized hippocampal injection of either artificial CSF (aCSF) or PT-3763 was performed (n=3). Hippocampal infusion of PT-3763 (1 M) led to a significantly larger suppression of activity in CA1, as compared to vehicle. D) Representative traces after localized administration of PT3763.


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Amperometry With the fiber photometry recordings, we were able to reproduce the ex vivo fEPSP Schaffer collateral effect. With amperometry we focused on assessing impact of PT-3763 on CA3 akin to the ex vivo associational connections. CA3 are glutamatergic pyramidal neurons 31, thus we hypothesized that PT-3763, through activation of presynaptic M4 mAChRs, will attenuate glutamate release in vivo. Through amperometry, glutamate release was quantified, on microsecond scale, in anesthetized rats where statistically significant effect (p = 0.02) of treatment with PT-3763 was observed (Figure 3A and 3B). Figure 3: M4 mAChR activation, through PT-3763 administration, attenuated hippocampal glutamate release. A) Representative trace of KCL-evoked glutamate release prior and following



local infusion of PT-3763. Arrow indicates the time of drug administration. B) Systemic administration of PT-3763 (32 mg/kg) led to significant attenuation of evoked glutamate release in CA3 as measured by amperometric probes (n = 8). Dashed line represents glutamate level prior to any treatment (vehicle or PT-3763).

A

0

Change from baseline (%)

B pre-PT-3763 32 mg/kg

glutamate (2 M)

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post-PT-3763 32 mg/kg

10

20

30

40

50

60

70

80

CA3 glutamate release

150

100 ------------------------------------------------

* 50

0

Vehicle

PT-3763

time (s)

Morris Water Maze (MWM) The ex vivo and in vivo electrophysiological and neurochemical changes observed with PT-3763 treatment led us to hypothesize that a behavioral effect, dependent on hippocampal circuitry, may present upon treatment with M4 mAChR PAM. Morris water maze is a spatial learning task that has been shown to rely on hippocampal CA3 associational connections 32 and to be adversely affected with scopolamine, a muscarinic receptor antagonist 33. Furthermore, donepezil, a cholinesterase inhibitor, was observed to reverse scopolamine-induced deficit in the Morris water maze 33,34 implicating the cholinergic system in this behavior. As reflected in the path length endpoint, scopolamine treated rats significantly underperformed vehicle treated rats (p < 0.0001) and the scopolamine-induced deficit was reversed with donepezil (p < 0.0001) (Figure 4A). Likewise, PT-3763 reversed the scopolamineinduced deficit across the three doses (0.32, 1 and 3.2 mg/kg) (p = 0.0173, p = 0.0063 and p = 0.0326, respectively; One-way ANOVA without assumption of equal variance p = 4.497e-10,


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overall main effect with false discovery rate (FDR) corrected post hoc analysis compared to Veh/Scop treatment group) (Figure 4A). Similarly, the escape latency measure also demonstrated a significant deficit induced by scopolamine (p

In vivo modulation of hippocampal excitability by M4 muscarinic

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