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Translational Utility of Mismatch Negativity: Implementing MMN in rats for Drug DiscoveryBackground: Mismatch negativity (MMN) is a validated, objective measure of central auditory processing and has become a key clinical biomarker in schizophrenia. MMN is an auditory evoked potential (AEP) elicited clinically by an auditory oddball paradigm in which a different, deviant (‘oddball’; DEV) auditory (tonal) stimulus occurs infrequently and unexpectedly within a sequence of repetitive...

S.C. LEISER1, D. BREGNA1, E. LEAHY1
1Translational EEG, PsychoGenics, Inc., Paramus, NJ, USA
Background Mismatch negativity (MMN) is a validated, objective measure of central auditory processing and has become a key clinical biomarker in schizophrenia. MMN is an auditory evoked potential (AEP) elicited clinically by an auditory oddball paradigm in which a different, deviant (‘oddball’; DEV) auditory (tonal) stimulus occurs infrequently and unexpectedly within a sequence of repetitive identical tonal stimuli (‘standards’, STD) and reflects pre-attentive processing dependent upon NMDA receptor function. Methods Here we demonstrate back-translatability of this AEP in Sprague-Dawley (SD) rats and show that NMDA receptor antagonism can impair rat MMN. First, we developed a system whereby we utilized the Data Sciences International (DSI) telemetry system to provide the flexibility and high-throughput nature of wireless recording up to 16 SD rats simultaneously in sound-attenuated chambers with the robust data handling and timestamping capabilities of the Cambridge Electronic Design (CED) micro1401 processor. A custom sequencer file enabled Spike2 software to generate tonal stimuli delivered to all rats simultaneously and timestamp the EEG data with digital precision. Rats were subjected to a standard oddball paradigm using 6kHz and 8kHz tones and assessed following MK-801 treatment. Results Vehicle-treated rats had a significantly larger response to DEV than STD as measured by peak and area under the curve components of the AEP waveform, while MK-801 (0.1-0.3 mg/kg) showed dose-dependent effects on impairing MMN. Further, in a manner consistent with clinical literature, the NMDA-antagonist induced impairment of rat MMN can be recovered by agents acting via NMDA receptor modulation. Moreover, an important recent finding in our laboratory is that, similar to findings in humans, rat AEPs are modulated of across vigilance states (sleep-wake). Discussion This work further validates that rats have a MMN correlate to that of the human MMN and that this biomarker is dependent upon similar mechanisms and receptor function. Further, we demonstrate the ability to perform this in a high-throughput manner necessary for drug discovery, which will lead to the further work needed to validate the translatability and predictive validity of the rat MMN.

Robust chemically induced animal model of Gaucher disease for preclinical studyParkinson’s disease (PD) and synucleinophathies are neurodegenerative disorders defined by α-synuclein (α-syn) accumulation. Mutations in the α-syn gene have been demonstrated in vitro and in vivo to accelerate the aggregation and formation of α-syn fibrils, a disease marker. Presence of α-syn positive Lewy bodies were identified in neuropathological analyses of a group of Gaucher disease type I [GD] patients [Sidransky, 2005]. Further human genetic studies.....

S. RAMBOZ, A. PENNINGTON, K. COX, M. HALL, W. ARIAS, K. KAYSER, J. GUTERL, M. BANSAL, D. HAVAS, K. CIRILLO
Parkinson’s disease (PD) and synucleinophathies are neurodegenerative disorders defined by α-synuclein (α-syn) accumulation. Mutations in the α-syn gene have been demonstrated in vitro and in vivo to accelerate the aggregation and formation of α-syn fibrils, a disease marker. Presence of α-syn positive Lewy bodies were identified in neuropathological analyses of a group of Gaucher disease type I [GD] patients [Sidransky, 2005]. Further human genetic studies have also linked glucocerebrosidase (GCase) gene GBA1 mutations to PD making this mutation the highest genetic risk factor to PD [Sidransky, 2009]. Due to the strong Robust chemically induced animal model of Gaucher disease for preclinical study clinical correlation between PD and GD diseases, animal models that demonstrate relevant and robust phenotypes [behavior, mRNA and protein profiling, IHC, ⋯] are of necessity. Genetically modified animals models carrying point mutations in GCase mimicking type 2 and type 3 have been generated targeting neuronal cells [Enquist et al. 2007; Liu et al. 1998]. In the present study, we focus on a chemically induced model consisting in daily injection of the irreversible GCase inhibitor conduritol B-epoxide (CBE) [Vardi et al., 2016). C57Bl6 mice were dosed daily via intraperitoneal injection at two different doses starting at post-natal day 8 to 22. Each pup was monitored closely and assess to motor, coordination and gait functions weekly from 3 weeks of age until study completion. Gait measures were assessed using PsychoGenics proprietary high through put gait platform, NeuroCube®. Preliminary assessment of data demonstrate progressive and CBE dose-dependent phenotypic deterioration of responses during assessments. Thus, our data demonstrate the progressive and robust phenotype of the chemically-induced GD model as an alternative to genetically modified animal models.

Behavioral assessment of the 61SNCA mouse model of Parkinson’s disease employing the NeuroCube® system of movement analysisParkinson’s disease (PD) is a neurodegenerative disorder characterized by dopaminergic neuronal loss in the substantia nigra with accumulation of α synuclein containing Lewy bodies. A number of rodent models of PD have been created to recapitulate different aspects of the disease, among these the Line 61 animal, overexpressing the human wild-type alpha-synuclein driven by the murine Thy-1 promoter, in particular has been used extensively to model α synuclein pathology [Rockenstein et al, 2002].

W. J. XU, A. EDWARDS, K. COX, K. CIRILLO, J. A. AVILA, S. RAMBOZ
Parkinson’s disease (PD) is a neurodegenerative disorder characterized by dopaminergic neuronal loss in the substantia nigra with accumulation of α synuclein containing Lewy bodies. A number of rodent models of PD have been created to recapitulate different aspects of the disease, among these the Line 61 animal, overexpressing the human wild-type alpha-synuclein driven by the murine Thy-1 promoter, in particular has been used extensively to model α synuclein pathology [Rockenstein et al, 2002]. While previous literature has characterized the male Line 61 mice and described a gradual disease progression, female counterparts of this model have been less well studied due to the linkage of the mutation to the X chromosome. In the present study, we performed a number of behavioral assessments in the female Line 61 including wire hang and tapered beam. We also employed a proprietary NeuroCube® assessment to evaluate quantifiable, subtle changes in patterned movement in these animals. This system records and calculates several aspects of ambulation and compares them to detect emerging movement impairments that may otherwise go unnoticed. Results suggest that a mild motor phenotype is present in the female Line 61 compared to both male Line 61 as well as WT counterparts. Implications of these findings as well as use of the NeuroCube® assessment as a tool in the evaluation of rodent models of disease are discussed.

Characterization of Htt-Q130-LEH, a novel heterozygous knock-in rat model of Huntington’s diseaseUsing a ZFN-mediated gene editing approach, a knock-in Long-Evans rat model of Huntington’s disease (Htt-Q130-LEH) was developed. The model carries a pure ∼130 CAG repeat followed by CAACAGCAGCAGCAACAG in Exon 1 of the endogenous rat Htt gene. The mHTT protein is expressed at ∼65% of the WT HTT protein throughout the CNS.

T. HEIKKINEN, T. BRAGGE, S. KOPONEN, T. PARKKARI, G. TOMBAUGH, S. GELMAN, S. ZHONG, L. DEVI, A. GHAVAMI, K. CIRILLO, A. BARRE, F. GACKIERE, D. GUIMOND, S. ZAPETTINI, B. SAVA, P. CHAUSSON, E. STEIDL, B. BUISSON, K. KUHLBRODT, A. KAKOULIDOU, S. JOBUSCH, K. TILLACK, A. GAERTNER, E. VAN DER KAM, R. Z. CHEN, D. HOWLAND, I. MUNOZ-SANJUAN, R. CACHOPE, J. ROSINSKI, V. BEAUMONT
Using a ZFN-mediated gene editing approach, a knock-in Long-Evans rat model of Huntington’s disease (Htt-Q130-LEH) was developed. The model carries a pure ∼130 CAG repeat followed by CAACAGCAGCAGCAACAG in Exon 1 of the endogenous rat Htt gene. The mHTT protein is expressed at ∼65% of the WT HTT protein throughout the CNS. Heterozygote (HET) and wild type (WT) littermates were subjected to a battery of motor tests every 3 months (m) starting at 3 m of age, including fine motor kinematic analysis, open field, tapered beam balance, grip strength, and weekly bodyweight monitoring. In addition, in vivo tetrode recordings of cortico-striatal and cortico-subthalamic nucleus (STN) transmission were assessed at 12 m for evidence of altered cortico-basal ganglia function. Ex vivo analysis included MesoScaleDiscovery (MSD) quantitation of mHTT, immunohistochemical (IHC) assessment of mHTT aggregation, RNA sequencing, and analysis of striatal medium spiny neuron (MSN) membrane properties and synaptic activity between 3 - 12 m of age. Until 6 months of age, HET Htt-Q130-LEH rats appear to be largely equivalent to their WT counterparts on all investigated parameters. However, with advancing age, HET rats start to display motor deficits, most notably as altered gait, decreased rearing and velocity in the open field. Bodyweight in male HET and WT rats is similar, whereas female HET rats are slightly heavier from 50 weeks onwards. At 12 m, in vivo electrophysiology showed that both cortico-striatal and cortico-STN transmission are significantly impaired in HET rats and striatal and cortical aggregates are detectable by MW8/4C9 MSD and S830 IHC analysis. Moreover, RNA sequencing revealed alterations in genes predominantly involved in GPCR / cAMP-mediated and calcium signaling, as well as neuro-inflammation. In summary, the Htt-Q130-LEH rat exhibits a relatively delayed onset of phenotype that will continue to be monitored at more advanced ages. This model may prove useful for further investigation of mHTT-induced neuropathophysiology.

Rare functional angiotensin I converting enzyme genetic variants increase the risk of Alzheimer’s diseaseThe angiotensin I converting enzyme (ACE1) gene (ACE) has been recently established as a genome-wide significant Alzheimer’s disease (AD) risk gene. ACE1 is a zinc-dependent peptidase that is known for regulating blood pressure within the circulatory renin-angiotensin system (RAS). However, ACE1 has diverse physiological functions, including a role in in the peripheral immune response, and an intrinsic RAS has been found in the brain where ACE1 is expressed in neurons.

L. K. CUDDY, D. PROKOPENKO, R. BRIMBERRY, E. CUNNINGHAM, P. SONG, D. PROCISSI, T. HANANIA, S. C. LEISER, R. E. TANZI, R. J. VASSAR
The angiotensin I converting enzyme (ACE1) gene (ACE) has been recently established as a genome-wide significant Alzheimer’s disease (AD) risk gene. ACE1 is a zinc-dependent peptidase that is known for regulating blood pressure within the circulatory renin-angiotensin system (RAS). However, ACE1 has diverse physiological functions, including a role in in the peripheral immune response, and an intrinsic RAS has been found in the brain where ACE1 is expressed in neurons. ACE1 has been previously linked to AD by observations that midlife hypertension increases AD risk, ACE1 degrades Aβ42 and ACE1 protein level is upregulated in post-mortem AD brains. In this study, we identify rare functional ACE variants that could help identify new biologic insights. Here we gained access to a deep (>40x) whole genome sequencing family-based cohort from NIMH. The dataset consisted of 446 families with affected and unaffected siblings. We further focused on medium and high impact rare variants. Among rare variants with a potential functional impact we selected rs4980 (R1279Q). In this study, we investigate ACE1 R1279Q in knock-in (ACE1KI/KI) mice with the cognate mutation in the murine ACE gene (R1284Q). We find that ACE1 R1284Q increases ACE1 protein level and activity in neurons and plasma, and is a toxic, gain-of-function mutation. ACE1KI/KI mice exhibited age-related neurodegeneration, EEG disruption and memory deficits, but had normal blood pressure and cerebrovascular function. In the brain, ACE1 R1284Q increased angiotensin II levels and caused selective neuron loss in the hippocampus. ACE1 R1284Q also increased the levels of circulating pro-inflammatory mediators known to be involved in the pathogenesis of autoimmune neuroinflammatory disorders. In 5XFAD ACE1KI/KI crosses, neurodegeneration was accelerated while Aβ42 level was unchanged. Finally, both central and peripherally-acting RAS inhibitors prevented neurodegeneration in ACE1KI/KI mice, suggesting phenotypes in ACE1KI/KI mice are caused by a combined effect of central and peripheral, blood pressure-independent actions of ACE1 R1284Q. Our findings support of growing evidence that RAS-acting anti-hypertensive medications may have beneficial effects toward the treatment or prevention of AD, independently of their blood pressure lowering properties or effects on Aβ42 clearance. Our studies show for the first time a direct link between ACE1 function, neurodegeneration and AD.
1Dept. of Neurol., 2Northwestern Univ., Chicago, IL; 3Gen Hosp, Harvard Med. Sch., Charlestown, MA; 4Northwestern Radiology, Chicago, IL; 5Translational EEG, PsychoGenics, Paramus, NJ.

SEP-363856, a novel psychotropic agent with a unique, non-D2 mechanism of actionFor the past 50 years, the clinical efficacy of antipsychotic medications has relied on blockade of dopamine D2 receptors. Drug development of non-D2 compounds, seeking to avoid the limiting side effects of dopamine blockade, has failed to date to yield new medicines for patients. Here we report the discovery of SEP-363856 (SEP-856), a novel psychotropic agent with a unique mechanism of action. SEP-856 was discovered in a medicinal chemistry effort utilizing a high throughput, high content, mouse-behavior phenotyping platform known as SmartCube®....

N. DEDIC1, P. G. JONES1, S. C. HOPKINS1, R. LEW1, T. HANANIA2, U. C. CAMPBELL1, K. S. KOBLAN1
1Sunovion Pharmaceuticals Inc., Marlborough, MA; 2Psychogenics Inc., Paramus, NJ.
For the past 50 years, the clinical efficacy of antipsychotic medications has relied on blockade of dopamine D2 receptors. Drug development of non-D2 compounds, seeking to avoid the limiting side effects of dopamine blockade, has failed to date to yield new medicines for patients. Here we report the discovery of SEP-363856 (SEP-856), a novel psychotropic agent with a unique mechanism of action. SEP-856 was discovered in a medicinal chemistry effort utilizing a high throughput, high content, mouse-behavior phenotyping platform known as SmartCube®, in combination with in vitro screening, aimed at developing non-D2 (anti-target) compounds that could nevertheless retain efficacy across a variety of D2-based animal models of schizophrenia. SEP-856 demonstrated broad efficacy in rodent models relating to positive- and negative symptoms of schizophrenia, including Phencyclidine (PCP)-induced hyperactivity, prepulse inhibition and PCP-induced deficits in social interaction. Autoradiography and positron emission tomography studies in rats and non-human primates demonstrated lack of D2 receptor occupancy by SEP-856 at concentrations up to 200-fold greater than those observed to be behaviorally efficacious. In addition to its favorable pharmacokinetic properties and the absence of catalepsy, SEP-856’s broad profile was further highlighted by its antidepressant activity in the mouse forced swim test and robust suppression of rapid eye movement sleep in the rat. Although, the mechanism of action has not been fully elucidated, in vitro and in vivo pharmacology data suggest that agonism at both 5-HT1A receptors and trace amine-associated receptor 1 (TAAR1) are integral to its efficacy. This was further corroborated with electrophysiological slice recordings, demonstrating inhibition of dorsal raphe nucleus and ventral tegmental area neuronal firing via 5-HT1A and TAAR1 receptors, respectively. Based on its unique mechanism of action and broad efficacy in preclinical animals models, SEP-856 was advanced into clinical development and represents a promising candidate for the treatment of schizophrenia and potentially other neuropsychiatric disorders.

The Michael J. Fox Foundation’s efforts to understand the relationship between GBA1 and alpha-synuclein through the development and characterization of preclinical modelsHeterozygous mutations in the GBA1 gene, which encodes lysosomal glucocerebrosidase (GCase), are the most common genetic risk factor for Parkinson’s disease (PD). In addition, decreased GCase activity has been reported in both genetic and sporadic cases of PD. Experimental evidence suggests a correlation between decreased GCase activity and accumulation of alpha-synuclein (aSyn). Thus, understanding the potential synergistic effect of increased aSyn and decreased GCase...

B. CASEY1, N. POLINSKI1, T. N. MARTINEZ1, S. W. CLARK2, S. M. SMITH3, R. C. SWITZER III4, S. O. AHMAD5, S. RAMBOZ6, M. SASNER7, M. T. HERBERTH8, L. MENALLED1, M. A. BAPTISTA1, K. D. DAVE1, M. FRASIER1
1The Michael J. Fox Fndn., New York, NY; 2Amicus Therapeut., Cranbury, NJ; 3Merck Res. Labs., West Point, PA; 4Neurosci. Associates Inc, Knoxville, TN; 5Doisy Hlth. Sciences: Office of Occup. Therapy, St. Louis Univ., Saint Louis, MO; 6Psychogenics Inc., Paramus, NJ; 7The Jackson Lab., Bar Harbor, ME; 8Charles River, Ashland, OH.
Heterozygous mutations in the GBA1 gene, which encodes lysosomal glucocerebrosidase (GCase), are the most common genetic risk factor for Parkinson’s disease (PD). In addition, decreased GCase activity has been reported in both genetic and sporadic cases of PD. Experimental evidence suggests a correlation between decreased GCase activity and accumulation of alpha-synuclein (aSyn). Thus, understanding the potential synergistic effect of increased aSyn and decreased GCase activity is important for understanding how alterations in GCase activity may contribute to or exacerbate PD-related pathology. To enable a better understanding of the relationship between aSyn and GCase activity, The Michael J. Fox Foundation (MJFF) has developed and characterized two mouse models allowing investigation of aSyn pathology in the context of reduced GCase activity. The first model analyzes the neurodegeneration/pathology induced through constitutive overexpression of wild type human alpha-synuclein directed by the murine Thy-1 promoter (hemizygous transgenic) in the context of the GCase activity-reducing D409V mutant form of GBA (homozygous knockin). The second model analyzes the level of nigrostriatal degeneration and synuclein pathology in the GBA D409V knockin model versus wildtype mice following stereotaxic injection of aSyn preformed fibrils into the striatum. Here, we outline and discuss the results of these model characterization efforts. Together, these models provide important platforms for understanding the mechanisms underlying GCase and aSyn dynamics, and for evaluating therapeutics targeting this pathway/relationship.

Differential impairment of cortico-striatal transmission onto direct and indirect pathway SPNs from Q175 miceChanges in corticostriatal neurotransmission have been identified as an early pathophysiological event observed in Huntington’s disease (HD), and model organisms have been a critical tool in furthering our understanding of these disease-driven processes. Examination of the Q175 mouse model of HD has uncovered changes in intrinsic membrane properties and excitability of both direct (D1 receptor-expressing) and indirect (D2 receptor-expressing) pathway medium spiny striatal projection neurons (SPNs).

H.B. FERNANDES1, J. SANCHEZ-PADILLA1, G. TOMBAUGH1, K. KRETSCHMANNOVA1, S. GELMAN1, A. GHAVAMI1, V. BEAUMONT2, R. CACHOPE2
1PsychoGenics, Inc., Paramus, NJ, USA; 2Translational Biology, CHDI Mgmt. / CHDI Foundation, Los Angeles, CA
Changes in corticostriatal neurotransmission have been identified as an early pathophysiological event observed in Huntington’s disease (HD), and model organisms have been a critical tool in furthering our understanding of these disease-driven processes. Examination of the Q175 mouse model of HD has uncovered changes in intrinsic membrane properties and excitability of both direct (D1 receptor-expressing) and indirect (D2 receptor-expressing) pathway medium spiny striatal projection neurons (SPNs). We have extended this work by probing the function of the direct and indirect corticostriatal pathways, using electrical stimulation of brain slices obtained from lines of Q175 mice expressing GFP in either D1-expressing (D1-GFP) or D2-expressing (D2-GFP) SPNs. We used three independent measures to assess excitatory presynaptic function: namely i) input-output relationship; ii) the relative size of the readily releasable pool (RRP) of synaptic vesicles; and iii) evoked Sr2+-mediated asynchronous release from cortical afferents. We found that glutamatergic corticostriatal transmission was decreased in indirect pathway (D2-GFP) SPNs, but was similar to WT in the direct (D1) pathway at the same age. This impairment in indirect pathway transmission was observed at several stimulus intensities. Paired pulse ratios at 25 ms and 50 ms intervals were unaffected in Q175het animals, suggesting that release probability was largely unaffected. Estimates of RRP size from a single stimulus train suggested a significant reduction in the number of vesicles available for release in indirect pathway corticostriatal terminals, while direct pathway terminals in Q175het slices were not significantly different from WT. We also observed a decrease in the frequency of asynchronous EPSC events following stimulation of indirect pathway Q175het corticostriatal afferents compared to WT, while no such deficit was observed in the direct pathway. These data are consistent with a corticostriatal deficit in neurotransmission in the indirect pathway of 6-month old Q175het mice, suggesting a potential pathway-specific mechanism of corticostriatal dysfunction.

Differential Patterns Of Microglial Reactivity In Accordance With Neurodegenerative Disease Types – A Comparative StudyObjectives: Today’s knowledge about microglial morphology and activation states vastly relies on in vitro cell culture experiments. Criticism to those theories rose steadily during the last decade, especially when looking at post mortem conditions in brain tissue. In Alzheimer’s disease (AD) animal models we see microglia clustering around amyloid plaques, de-ramifying and with increased somal size.

D. HAVAS1, J. PINDJAKOVA, I. MORGANSTERN1, T. HANANIA1
1PsychoGenics, Inc., Paramus, NJ, USA
Objectives: Today’s knowledge about microglial morphology and activation states vastly relies on in vitro cell culture experiments. Criticism to those theories rose steadily during the last decade, especially when looking at post mortem conditions in brain tissue. In Alzheimer’s disease (AD) animal models we see microglia clustering around amyloid plaques, de-ramifying and with increased somal size. In frontotemporal lobe dementia (FTLD) and Tauopathy model Tg4510, we see cytosis paired with macrophagicity (CD68 positive) in any kind of morphological state, while somal size is smaller and cells are not re-ramified at late stage. In amyotrophic lateral sclerosis (SOD1G93A), we found a 40-fold increase of large ramifying microglia with strong de-ramification. Last but not least, Rett syndrome mice with immune deficiency seem to lose the ability to change ramification and both small and large ramifying cells are significantly reduced. All those findings do not fit into the classical understanding of microglial reactivity as found in cell culture.
Methods: We developed a tool set for histological post mortem quantification of microglial morphology. This set includes classical Iba1 counting widened with type classification by computer learned algorithms and additional structural analysis of somal size, branching and process lengths. Together those data define a pattern of microglial reactivity.
Results: Finally we found out that we are able to distinguish the type of disease by pattern match of microglial alterations in Rett syndrome, AD, FTLD, Tauopathy and ALS models measured in controls of a series of preclinical studies.
Conclusions: Those data suggest to rethink the hitherto theory for ex vivo work and rather in first line to compare these patterns with those found in human disease samples and in second, to find out which mechanisms are responsible for the diversity of microglial reactivity patterns in different disease types.