Inositol Phosphate Accumulation in Vivo Provides a Measure of Muscarinic M₁ Receptor Activation

The rationale for using M₁ selective muscarinic acetylcholine receptor activators for the treatment of cognitive impairment associated with psychiatric and neurodegenerative disease is well-established in the literature. Here, we investigate measurement of inositol phosphate accumulation, an end point immediately downstream of the M₁ muscarinic acetylcholine receptor signaling cascade, as an in vivo biochemical readout for M₁ muscarinic acetylcholine receptor activation. Five brain penetrant M₁-subtype selective activators from three structurally distinct chemical series were pharmacologically profiled for functional activity in vitro using recombinant cell calcium mobilization and inositol phosphate assays, and a native tissue hippocampal slice electrophysiology assay, to show that all five compounds presented a positive allosteric modulator agonist profile, within a narrow range of potencies. In vivo characterization using an amphetamine-stimulated locomotor activity behavioral assay and the inositol phosphate accumulation biochemical assay demonstrated that the latter has utility for assessing functional potency of M₁ activators. Efficacy measured by inositol phosphate accumulation in mouse striatum compared favorably to efficacy in reversing amphetamine-induced locomotor activity, suggesting that the inositol phosphate accumulation assay has utility for the evaluation of M₁ muscarinic acetylcholine receptor activators in vivo. The benefits of this in vivo biochemical approach include a wide response window, interrogation of specific brain circuit activation, an ability to model responses in the context of brain exposure, an ability to rank order compounds based on in vivo efficacy, and minimization of animal use

Behavioral Characterization of A53T Mice Reveals Early and Late Stage Deficits Related to Parkinson’s Disease

<div><p>Parkinson's disease (PD) pathology is characterized by the formation of intra-neuronal inclusions called Lewy bodies, which are comprised of alpha-synuclein (α-syn). Duplication, triplication or genetic mutations in α-syn (A53T, A30P and E46K) are linked to autosomal dominant PD; thus implicating its role in the pathogenesis of PD. In both PD patients and mouse models, there is increasing evidence that neuronal dysfunction occurs before the accumulation of protein aggregates (i.e., α-syn) and neurodegeneration. Characterization of the timing and nature of symptomatic dysfunction is important for understanding the impact of α-syn on disease progression. Furthermore, this knowledge is essential for identifying pathways and molecular targets for therapeutic intervention. To this end, we examined various functional and morphological endpoints in the transgenic mouse model expressing the human A53T α-syn variant directed by the mouse prion promoter at specific ages relating to disease progression (2, 6 and 12 months of age). Our findings indicate A53T mice develop fine, sensorimotor, and synaptic deficits before the onset of age-related gross motor and cognitive dysfunction. Results from open field and rotarod tests show A53T mice develop age-dependent changes in locomotor activity and reduced anxiety-like behavior. Additionally, digigait analysis shows these mice develop an abnormal gait by 12 months of age. A53T mice also exhibit spatial memory deficits at 6 and 12 months, as demonstrated by Y-maze performance. In contrast to gross motor and cognitive changes, A53T mice display significant impairments in fine- and sensorimotor tasks such as grooming, nest building and acoustic startle as early as 1–2 months of age. These mice also show significant abnormalities in basal synaptic transmission, paired-pulse facilitation and long-term depression (LTD). Combined, these data indicate the A53T model exhibits early- and late-onset behavioral and synaptic impairments similar to PD patients and may provide useful endpoints for assessing novel therapeutic interventions for PD.</p></div

Synaptic deficits present in the CA1 region of A53T mice as early as 2 months.

<p>Field potentials were evoked by stimulating the Schaffer collaterals with a concentric bipolar electrode and recorded with glass electrodes in the stratum radiatum of CA1. Basal synaptic transmission is significantly impaired in 2 month A53T mice compared to wild type littermates (A). Paired-pulse facilitation (PPF) is significantly enhanced in 2 month-old A53T mice (B). Theta burst stimulation (TBS) induces LTP similarly in 2 month-old wild type and A53T mice (C). Low frequency stimulation (LFS, 900 pulses at 1 Hz) induces short and long term depression in wild type mice; however, it induces both short and long term potentiation in A53T mice (D). Data plotted as Mean+/− SEM. fEPSP = Field Excitatory Post-Synaptic Potentials, mV = millivolts, min = minutes.</p

A53T mice develop a reduced anxiety–like phenotype with age.

<p>Mice were allowed to freely explore a brightly lit open field for 60 minutes. Graph shows the total time spent in the center of the field from 2-, 6- and 12-month wild type and A53T mice (A). A53T mice spend significantly more time in the center of the open field at 12 months of age compared to wild type littermates, suggesting a reduced anxiety–like phenotype. The initial body temperature (T1) was recorded via a rectal probe (°C), then a second rectal temperature (T2) is recorded 10 minutes following T1. The stress of the initial rectal temperature recording induces an endogenous increase in body temperature known as the stress-induced hyperthermic response SIH = ΔT (T2°–T1°). Graphs show the hyperthermic response from 2-, 6- and 12-month wild type and A53T mice (B). Although not statistically significant (p = 0.177), there was a trend for an attenuated Stress-Induced hyperthermic (SIH) response relative to wild type control in 12 month old mice, which may be indicative of an anxiolytic-like phenotype. Graphs are representative of data collected from 2–3 independent cohorts of animals with 8–15 mice per group. Bonferonni post hoc test ***p<0.0001; WT = Wild type, HO = Homozygous, M = Month, sec = seconds, T1 = Test 1, T2 = Test 2, C = Celsius.</p

Total and aggregated mutant alpha synuclein (α-syn) increases with age and onset of disease.

<p>Human specific α-syn was expressed in neurons and neuropil of A53T homozygote (top panel), but not wild type mice (bottom panel; A). Western blot analysis indicates human α-syn levels significantly increase with age in A53T brain (n = 6 per group; B). α-syn aggregates are rare but visible at 2 months, then accumulate with age; representative photo of proteinase-K resistant aggregates in A53T brain (pons region) at 2, 6 and 12 months (C). Kaplan-Meier survival curve illustrates onset of disease, as defined by motor disability, between 8–17 months in homozygous A53T mice (n = 30; D). Data normalized to actin and plotted as Mean+/− SEM. **p<0.001.</p

A53T mice exhibit age-related spatial memory deficits.

<p>Mice were allowed to explore two arms (start & familiar) of the Y-maze for five minutes (Trial 1). During this acclimation period, both wild type and A53T mice performed similarly when exploring (A) and entering (B) arms. The percent duration in each arm for 2 month old wild type mice was different than chance (#, 95% confidence interval; dotted line = 50%); however the overall entries and total time spent in arms were similar for both genotypes (data not shown). After a one-hour inter-trial interval, wild type mice of all ages and 2-month-old A53T mice spent significantly more time exploring (C) and entering (D) the novel arm of the Y-maze compared to the start or familiar arms. Conversely, 6 and 12 month old A53T mice did not show a preference for the novel arm, suggesting a disruption in spatial memory. There were no alterations in locomotor indices across genotype or age (not shown). Data plotted as Mean +/−SEM. To look for equal exploration of arms, data were compared to chance (depicted by dotted line; T1 = 50%, T2 = 33.3%) using the 95% confidence interval (^#p<0.05). Unpaired t-test analysis was utilized for assessing differences between genotypes within age (*p<0.05). n = 11–15 per group; W = wild type, H = homozygote.</p

A53T mice elicit a reduced startle response at 2 months of age.

<p>The startle reflex response is significantly reduced in homozygous A53T mice relative to wild type controls at 2, 6 and 12 months (A). Results from the auditory brainstem response test suggest the reduction in startle is not due to hearing loss as there is no difference between wild type and A53T mice in hearing when exposed to frequencies of 8 and 16 kHz. In fact, A53T mice actually hear at a lower threshold when exposed to the 32 kHz frequency (N = 6 per group; 2 months of age) (B). Unless otherwise noted, graphs are representative of data collected from 2–3 independent cohorts of animals with 8–15 mice per group. Data plotted as Mean+/− SEM. Bonferonni post hoc test *p<0.05; **p<0.001; ***p<0.0001; kHz = kilohertz, dB = decibel, WT = Wild type, HO = Homozygous.</p

A53T homozygote mice develop gross motor abnormalities with age.

<p>In the open field, results from spontaneous locomotor testing indicate A53T mice travel significantly greater distances than wild type littermates at 6 and 12 months (A). Digigait analysis shows the stride length and frequency (steps per second) are significantly decreased in 12 month A53T mice compared to wild type littermates (t-test analysis within age group- data represent both hind limbs (B). The combined latency to fall from 5 individual trials shows A53T mice exhibit enhanced rotarod latency that declines with age (C). Graphs are representative of data collected from 2–3 independent cohorts of animals with 8–15 mice per group. Data plotted as Mean+/− SEM. Bonferonni post hoc test *p<0.05, **p<0.001; s = seconds, cm = centimeters, M = month, WT = wild type, HO = homozygous.</p