Neuronopathic GBA1L444P Mutation Accelerates Glucosylsphingosine Levels and Formation of Hippocampal Alpha-Synuclein Inclusions.

The most common genetic risk factor for Parkinson's disease (PD) is heterozygous mutations GBA1, which encodes for the lysosomal enzyme, glucocerebrosidase. Reduced glucocerebrosidase activity associates with an accumulation of abnormal α-synuclein (α-syn) called Lewy pathology, which characterizes PD. PD patients heterozygous for the neuronotypic GBA1L444P mutation (GBA1+/L444P) have a 5.6-fold increased risk of cognitive impairments. In this study, we used GBA1+/L444P mice of either sex to determine its effects on lipid metabolism, expression of synaptic proteins, behavior, and α-syn inclusion formation. At 3 months of age, GBA1+/L444P mice demonstrated impaired contextual fear conditioning, and increased motor activity. Hippocampal levels of vGLUT1 were selectively reduced in GBA1+/L444P mice. We show, using mass spectrometry, that GBA1L444P expression increased levels of glucosylsphingosine, but not glucosylceramide, in the brains and serum of GBA1+/L444P mice. Templated induction of α-syn pathology in mice showed an increase in α-syn inclusion formation in the hippocampus of GBA1+/L444P mice compared with GBA1+/+ mice, but not in the cortex, or substantia nigra pars compacta. Pathologic α-syn reduced SNc dopamine neurons by 50% in both GBA1+/+ and GBA1+/L444P mice. Treatment with a GlcCer synthase inhibitor did not affect abundance of α-syn inclusions in the hippocampus or rescue dopamine neuron loss. Overall, these data suggest the importance of evaluating the contribution of elevated glucosylsphingosine to PD phenotypes. Further, our data suggest that expression of neuronotypic GBA1L444P may cause defects in the hippocampus, which may be a mechanism by which cognitive decline is more prevalent in individuals with GBA1-PD.SIGNIFICANCE STATEMENT Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are both pathologically characterized by abnormal α-synuclein (α-syn). Mutant GBA1 is a risk factor for both PD and DLB. Our data show the expression of neuronotypic GBA1L444P impairs behaviors related to hippocampal function, reduces expression of a hippocampal excitatory synaptic protein, and that the hippocampus is more susceptible to α-syn inclusion formation. Further, our data strengthen support for the importance of evaluating the contribution of glucosylsphingosine to PD phenotypes. These outcomes suggest potential mechanisms by which GBA1L444P contributes to the cognitive symptoms clinically observed in PD and DLB. Our findings also highlight the importance of glucosylsphingosine as a relevant biomarker for future therapeutics.

Validation of a multiplexed and targeted lipidomics assay for accurate quantification of lipidomes.

A major challenge of lipidomics is to determine and quantify the precise content of complex lipidomes to the exact lipid molecular species. Often, multiple methods are needed to achieve sufficient lipidomic coverage to make these determinations. Multiplexed targeted assays offer a practical alternative to enable quantitative lipidomics amenable to quality control standards within a scalable platform. Herein, we developed a multiplexed normal phase liquid chromatography-hydrophilic interaction chromatography multiple reaction monitoring method that quantifies lipid molecular species across over 20 lipid classes spanning wide polarities in a single 20-min run. Analytical challenges such as in-source fragmentation, isomer separations, and concentration dynamics were addressed to ensure confidence in selectivity, quantification, and reproducibility. Utilizing multiple MS/MS product ions per lipid species not only improved the confidence of lipid identification but also enabled the determination of relative abundances of positional isomers in samples. Lipid class-based calibration curves were applied to interpolate lipid concentrations and guide sample dilution. Analytical validation was performed following FDA Bioanalytical Method Validation Guidance for Industry. We report repeatable and robust quantitation of 900 lipid species measured in NIST-SRM-1950 plasma, with over 700 lipids achieving inter-assay variability below 25%. To demonstrate proof of concept for biomarker discovery, we analyzed plasma from mice treated with a glucosylceramide synthase inhibitor, benzoxazole 1. We observed expected reductions in glucosylceramide levels in treated animals but, more notably, identified novel lipid biomarker candidates from the plasma lipidome. These data highlight the utility of this qualified lipidomic platform for enabling biological discovery.

A novel glucosylceramide synthase inhibitor attenuates alpha synuclein pathology and lysosomal dysfunction in preclinical models of synucleinopathy.

Mutations in the lysosomal enzyme glucocerebrosidase (GCase, GBA1 gene) are the most common genetic risk factor for developing Parkinson's disease (PD). GCase metabolizes the glycosphingolipids glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). Mutations in GBA1 reduce enzyme activity and the resulting accumulation of glycosphingolipids may contribute to the underlying pathology of PD, possibly via altering lysosomal function. While reduction of GCase activity exacerbates α-synuclein (α-syn) aggregation, it has not been determined that this effect is the result of altered glycosphingolipid levels and lysosome function or some other effect of altering GCase. The glycosphingolipid GlcCer is synthesized by a single enzyme, glucosylceramide synthase (GCS), and small molecule inhibitors (GCSi) reduce cellular glycosphingolipid levels. In the present studies, we utilize a preformed fibril (PFF) rodent primary neuron in vitro model of α-syn pathology to investigate the relationship between glycosphingolipid levels, α-syn pathology, and lysosomal function. In primary cultures, pharmacological inhibition of GCase and D409V GBA1 mutation enhanced accumulation of glycosphingolipids and insoluble phosphorylated α-syn. Administration of a novel small molecule GCSi, benzoxazole 1 (BZ1), significantly decreased glycosphingolipid concentrations in rodent primary neurons and reduced α-syn pathology. BZ1 rescued lysosomal deficits associated with the D409V GBA1 mutation and α-syn PFF administration, and attenuated α-syn induced neurodegeneration of dopamine neurons. In vivo studies revealed BZ1 had pharmacological activity and reduced glycosphingolipids in the mouse brain to a similar extent observed in neuronal cultures. These data support the hypothesis that reduction of glycosphingolipids through GCS inhibition may impact progression of synucleinopathy and BZ1 is useful tool to further examine this important biology.

Orexin receptor 2 expression in the posterior hypothalamus rescues sleepiness in narcoleptic mice.

Narcolepsy is caused by a loss of orexin/hypocretin signaling, resulting in chronic sleepiness, fragmented non-rapid eye movement sleep, and cataplexy. To identify the neuronal circuits underlying narcolepsy, we produced a mouse model in which a loxP-flanked gene cassette disrupts production of the orexin receptor type 2 (OX2R; also known as HCRTR2), but normal OX2R expression can be restored by Cre recombinase. Mice lacking OX2R signaling had poor maintenance of wakefulness indicative of sleepiness and fragmented sleep and lacked any electrophysiological response to orexin-A in the wake-promoting neurons of the tuberomammillary nucleus. These defects were completely recovered by crossing them with mice that express Cre in the female germline, thus globally deleting the transcription-disrupter cassette. Then, by using an adeno-associated viral vector coding for Cre recombinase, we found that focal restoration of OX2R in neurons of the tuberomammillary nucleus and adjacent parts of the posterior hypothalamus completely rescued the sleepiness of these mice, but their fragmented sleep was unimproved. These observations demonstrate that the tuberomammillary region plays an essential role in the wake-promoting effects of orexins, but orexins must stabilize sleep through other targets.

Fluorinated Isoindolinone-Based Glucosylceramide Synthase Inhibitors with Low Human Dose Projections.

Inhibition of glucosylceramide synthase (GCS) has been proposed as a therapeutic strategy for the treatment of Parkinson's Disease (PD), particularly in patients where glycosphingolipid accumulation and lysosomal impairment are thought to be contributing to disease progression. Herein, we report the late-stage optimization of an orally bioavailable and CNS penetrant isoindolinone class of GCS inhibitors. Starting from advanced lead 1, we describe efforts to identify an improved compound with a lower human dose projection, minimal P-glycoprotein (P-gp) efflux, and acceptable pregnane X receptor (PXR) profile through fluorine substitution. Our strategy involved the use of predicted volume ligand efficiency to advance compounds with greater potential for low human doses down our screening funnel. We also applied minimized electrostatic potentials (Vmin) calculations for hydrogen bond acceptor sites to rationalize P-gp SAR. Together, our strategies enabled the alignment of a lower human dose with reduced P-gp efflux, and favorable PXR selectivity for the discovery of compound 12.

Pyrazole Ureas as Low Dose, CNS Penetrant Glucosylceramide Synthase Inhibitors for the Treatment of Parkinson's Disease.

Parkinson's disease is the second most prevalent progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra. Loss-of-function mutations in GBA, the gene that encodes for the lysosomal enzyme glucosylcerebrosidase, are a major genetic risk factor for the development of Parkinson's disease potentially through the accumulation of glucosylceramide and glucosylsphingosine in the CNS. A therapeutic strategy to reduce glycosphingolipid accumulation in the CNS would entail inhibition of the enzyme responsible for their synthesis, glucosylceramide synthase (GCS). Herein, we report the optimization of a bicyclic pyrazole amide GCS inhibitor discovered through HTS to low dose, oral, CNS penetrant, bicyclic pyrazole urea GCSi's with in vivo activity in mouse models and ex vivo activity in iPSC neuronal models of synucleinopathy and lysosomal dysfunction. This was accomplished through the judicious use of parallel medicinal chemistry, direct-to-biology screening, physics-based rationalization of transporter profiles, pharmacophore modeling, and use a novel metric: volume ligand efficiency.

The GBA1 D409V mutation exacerbates synuclein pathology to differing extents in two alpha-synuclein models.

Heterozygous mutations in the GBA1 gene - encoding lysosomal glucocerebrosidase (GCase) - are the most common genetic risk factors for Parkinson's disease (PD). Experimental evidence suggests a correlation between decreased GCase activity and accumulation of alpha-synuclein (aSyn). To enable a better understanding of the relationship between aSyn and GCase activity, we developed and characterized two mouse models that investigate aSyn pathology in the context of reduced GCase activity. The first model used constitutive overexpression of wild-type human aSyn in the context of the homozygous GCase activity-reducing D409V mutant form of GBA1. Although increased aSyn pathology and grip strength reductions were observed in this model, the nigrostriatal system remained largely intact. The second model involved injection of aSyn preformed fibrils (PFFs) into the striatum of the homozygous GBA1 D409V knock-in mouse model. The GBA1 D409V mutation did not exacerbate the pathology induced by aSyn PFF injection. This study sheds light on the relationship between aSyn and GCase in mouse models, highlighting the impact of model design on the ability to model a relationship between these proteins in PD-related pathology.

Intracranial administration of alpha-synuclein fibrils in A30P-synuclein transgenic mice causes robust synucleinopathy and microglial induction.

Synucleinopathies are neurodegenerative disorders involving pathological alpha-synuclein (αSyn) protein, including dementia with Lewy bodies, multiple system atrophy and Parkinson's disease (PD). Current in vivo models of synucleinopathy include transgenic mice overexpressing αSyn variants and methods based on administration of aggregated, exogenous αSyn. Combining these techniques offers the ability to study consequences of introducing pathological αSyn into primed neuronal environments likely to develop synucleinopathy. Herein, we characterize the impacts pre-formed fibrils (PFFs) of recombinant, human αSyn have in mice overexpressing human A30P αSyn, a mutation associated with autosomal dominant PD. A30P mouse brain contains detergent insoluble αSyn biochemically similar to PD brain, and these mice develop Lewy-like synucleinopathy with age. Administration of PFFs in A30P mice resulted in regionally-specific accumulations of phosphorylated synuclein, microglial induction and a motor phenotype that differed from PFF-induced effects in wildtype mice. Surprisingly, PFF-induced losses of tyrosine hydroxylase were similar in A30P and wildtype mice. Thus, the PFF-A30P model recapitulates key aspects of synucleinopathy with induction of microglia, creating an appropriate system for evaluating neurodegenerative therapeutics.

Orexin neurons and the TASK of glucosensing.

Relatively little is known about the mechanisms that link changing levels of glucose and neuronal activity. A paper in the current issue of Neuron by Burdakov et al. demonstrates that orexin/hypocretin neurons are inhibited by rising glucose in part due to membrane potential effects mediated by tandem-pore K(+) (K(2P)) channels. The findings may shed light on the mechanisms that link hypoglycemia and coordinated arousal and autonomic responses.

Distinct narcolepsy syndromes in Orexin receptor-2 and Orexin null mice: molecular genetic dissection of Non-REM and REM sleep regulatory processes.

Narcolepsy-cataplexy, a neurological disorder associated with the absence of hypothalamic orexin (hypocretin) neuropeptides, consists of two underlying problems: inability to maintain wakefulness and intrusion of rapid eye movement (REM) sleep into wakefulness. Here we document, using behavioral, electrophysiological, and pharmacological criteria, two distinct classes of behavioral arrests exhibited by mice deficient in orexin-mediated signaling. Both OX2R(-/-) and orexin(-/-) mice are similarly affected with behaviorally abnormal attacks of non-REM sleep ("sleep attacks") and show similar degrees of disrupted wakefulness. In contrast, OX2R(-/-) mice are only mildly affected with cataplexy-like attacks of REM sleep, whereas orexin(-/-) mice are severely affected. Absence of OX2Rs eliminates orexin-evoked excitation of histaminergic neurons in the hypothalamus, which gate non-REM sleep onset. While normal regulation of wake/non-REM sleep transitions depends critically upon OX2R activation, the profound dysregulation of REM sleep control unique to the narcolepsy-cataplexy syndrome emerges from loss of signaling through both OX2R-dependent and OX2R-independent pathways.

Mice lacking ghrelin receptors resist the development of diet-induced obesity.

Ghrelin is the endogenous ligand for the growth hormone secretagogue receptor (GHSR; ghrelin receptor). Since its discovery, accumulating evidence has suggested that ghrelin may play a role in signaling and reversing states of energy insufficiency. For example, ghrelin levels rise following food deprivation, and ghrelin administration stimulates feeding and increases body weight and adiposity. However, recent loss-of-function studies have raised questions regarding the physiological significance of ghrelin in regulating these processes. Here, we present results of a study using a novel GHSR-null mouse model, in which ghrelin administration fails to acutely stimulate food intake or activate arcuate nucleus neurons. We show that when fed a high-fat diet, both female and male GHSR-null mice eat less food, store less of their consumed calories, preferentially utilize fat as an energy substrate, and accumulate less body weight and adiposity than control mice. Similar effects on body weight and adiposity were also observed in female, but not male, GHSR-null mice fed standard chow. GHSR deletion also affected locomotor activity and levels of glycemia. These findings support the hypothesis that ghrelin-responsive pathways are an important component of coordinated body weight control. Moreover, our data suggest that ghrelin signaling is required for development of the full phenotype of diet-induced obesity.

Excitotoxic lesions restricted to the dorsal CA1 field of the hippocampus impair spatial memory and extinction learning in C57BL/6 mice.

Recent studies in patients with hippocampal lesions have indicated that the degree of memory impairment is proportional to the extent of damage within the hippocampus. Particularly, patients with damage restricted to the CA1 field demonstrate moderate to severe anterograde amnesia with only slight retrograde amnesia. Comparable results are also seen in other species such as non-human primates and rats; however, the effect of selective damage to CA1 has not yet been characterized in mice. In the present study, we investigated the effects of excitotoxic (NMDA) lesions of dorsal CA1 on several aspects of learning and memory performance in mice. Our data indicate that dorsal CA1 lesioned mice are hyperactive upon exposure to a novel environment, have spatial working memory impairments in the Y-maze spontaneous alternation task, and display deficits in an 8-arm spatial discrimination learning task. Lesioned mice are able to acquire an operant lever-press task but demonstrate extinction learning deficits in this appetitive operant paradigm. Taken together, our results indicate that lesions to dorsal CA1 in mice induce selective learning and memory performance deficits similar to those observed in other species, and extend previous findings indicating that this region of the hippocampus is critically involved in the processing of spatial information and/or the processing of inhibitory responses.

Glucagon-like peptide-1 receptor stimulation increases blood pressure and heart rate and activates autonomic regulatory neurons.

Glucagon-like peptide-1 (GLP-1) released from the gut functions as an incretin that stimulates insulin secretion. GLP-1 is also a brain neuropeptide that controls feeding and drinking behavior and gastric emptying and elicits neuroendocrine responses including development of conditioned taste aversion. Although GLP-1 receptor (GLP-1R) agonists are under development for the treatment of diabetes, GLP-1 administration may increase blood pressure and heart rate in vivo. We report here that centrally and peripherally administered GLP-1R agonists dose-dependently increased blood pressure and heart rate. GLP-1R activation induced c-fos expression in the adrenal medulla and neurons in autonomic control sites in the rat brain, including medullary catecholamine neurons providing input to sympathetic preganglionic neurons. Furthermore, GLP-1R agonists rapidly activated tyrosine hydroxylase transcription in brainstem catecholamine neurons. These findings suggest that the central GLP-1 system represents a regulator of sympathetic outflow leading to downstream activation of cardiovascular responses in vivo.

Hcrtr1 and 2 signaling differentially regulates depression-like behaviors.

The orexin/hypocretin system has the potential to significantly modulate affect, based on both the neuroanatomical projection patterns of these neurons and on the sites of orexin receptor expression. However, there is little data supporting the role of specific orexin receptors in the modulation of depression-like behavior. Here we report behavioral profiling of mice after genetic or pharmacologic inhibition of hcrtr1 and 2 receptor signaling. Hcrtr1 null mice displayed a significant reduction in behavioral despair in the forced swim test and tail suspension test. Wild-type mice treated with the hcrtr1 antagonist SB-334867 also displayed a similar reduction in behavioral despair. No difference in anxiety-like behavior was noted following hcrtr1 deletion. In contrast, hcrtr2-null mice displayed an increase in behavioral despair with no effect on measures of anxiety. These studies suggest that the balance of orexin action at either the hcrtr1 or the hcrtr2 receptor produces an anti-depressant or pro-depressant like effect, depending on the receptor subtype activated.

Prefrontal cortex lesions and scopolamine impair attention performance of C57BL/6 mice in a novel 2-choice visual discrimination task.

Sustained attention is defined as the ability or capacity to remain focused on the occurrence of rare events over long periods of time. We describe here the development of a novel, operant-based attention task that can be learned by mice in 8-10 days. Mice were trained on a 2-choice visual discrimination task in an operant chamber, wherein the correct response on any given trial was a lever-press cued by a stimulus light. Upon reaching a criterion of greater than 80% correct responses, all subjects were tested in a mixed-trial attention paradigm combining four different stimulus durations within a single session (0.5, 1, 2, or 10 s). During attention testing, the percentage of correct responses decreased as a function of stimulus duration, indicating a performance decrement which parallels increasing attentional demand within the task. Pretreatment with the muscarinic-receptor antagonist scopolamine yielded a reliable, dose-dependent performance deficit whereas nicotine treatment improved the percentage of correct responses during trials with the greatest attentional demand. Moreover, medial prefrontal cortex lesions impaired attention performance without affecting acquisition or retention of the discrimination rule. These results underscore the utility of this task as a novel means of assessing attentional processes in mice in a relatively high-throughput manner.

Activation of central melanocortin pathways by fenfluramine.

D-fenfluramine (d-FEN) was once widely prescribed and was among the most effective weight loss drugs, but was withdrawn from clinical use because of reports of cardiac complications in a subset of patients. Discerning the neurobiology underlying the anorexic action of d-FEN may facilitate the development of new drugs to prevent and treat obesity. Through a combination of functional neuroanatomy, feeding, and electrophysiology studies in rodents, we show that d-FEN-induced anorexia requires activation of central nervous system melanocortin pathways. These results provide a mechanistic explanation of d-FEN's anorexic actions and indicate that drugs targeting these downstream melanocortin pathways may prove to be effective and more selective anti-obesity treatments.