Taylor Family Institute for Innovative Psychiatric Research

The recent FDA approval of the neurosteroid, brexanolone (allopregnanolone), as a treatment for women with postpartum depression, and successful trials of a related neuroactive steroid, SGE-217, for men and women with major depressive disorder offer the hope of a new era in treating mood and anxiety disorders based on the potential of neurosteroids as modulators of brain function. This review considers potential mechanisms contributing to antidepressant and anxiolytic effects of allopregnanolone and other GABAergic neurosteroids focusing on their actions as positive allosteric modulators of GABAA receptors. We also consider their roles as endogenous “stress” modulators and possible additional mechanisms contributing to their therapeutic effects. We argue that further understanding of the molecular, cellular, network and psychiatric effects of neurosteroids offers the hope of further advances in the treatment of mood and anxiety disorders.

N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels that are critical to the regulation of excitatory synaptic function in the CNS. NMDARs govern experience-dependent synaptic plasticity and have been implicated in the pathophysiology of various neuropsychiatric disorders including the cognitive deficits of schizophrenia and certain forms of autism. Certain neurosteroids modulate NMDARs experimentally but their low potency, poor selectivity, and very low brain concentrations make them poor candidates as endogenous ligands or therapeutic agents. Here we show that the major brain-derived cholesterol metabolite 24(S)-hydroxycholesterol (24(S)-HC) is a very potent, direct, and selective positive allosteric modulator of NMDARs with a mechanism that does not overlap that of other allosteric modulators. At submicromolar concentrations 24(S)-HC potentiates NMDAR-mediated EPSCs in rat hippocampal neurons but fails to affect AMPAR or GABAA receptors (GABA(A)Rs)-mediated responses. Cholesterol itself and other naturally occurring oxysterols present in brain do not modulate NMDARs at concentrations ≤10 μM. In hippocampal slices, 24(S)-HC enhances the ability of subthreshold stimuli to induce long-term potentiation (LTP). 24(S)-HC also reverses hippocampal LTP deficits induced by the NMDAR channel blocker ketamine. Finally, we show that synthetic drug-like derivatives of 24(S)-HC, which potently enhance NMDAR-mediated EPSCs and LTP, restore behavioral and cognitive deficits in rodents treated with NMDAR channel blockers. Thus, 24(S)-HC may function as an endogenous modulator of NMDARs acting at a novel oxysterol modulatory site that also represents a target for therapeutic drug development.

Human studies examining the effects of the dissociative anesthetic ketamine as a model for psychosis and as a rapidly acting antidepressant have spurred great interest in understanding ketamine's actions at molecular, cellular, and network levels. Although ketamine has unequivocal uncompetitive inhibitory effects on N-methyl-d-aspartate receptors (NMDARs) and may preferentially alter the function of NMDARs on interneurons, recent work has questioned whether block of NMDARs is critical for its mood enhancing actions. In this viewpoint, we examine the evolving literature on ketamine supporting NMDARs as important triggers for certain psychiatric effects and the possibility that the antidepressant trigger is unrelated to NMDARs. The rapidly evolving story of ketamine offers great hope for untangling and treating the biology of both depressive and psychotic illnesses.

OBJECTIVE: We previously found a strong reduction in tau pathology and insoluble tau in P301S tau transgenic mice following intracerebroventricular infusion of the anti-tau antibody HJ8.5. We sought to determine the effects of HJ8.5 in the same model following peripheral administration. METHODS: The primary objective was to determine if HJ8.5 administered at a dose of 50 mg kg(-1) week(-1) by intraperitoneal (IP) injection to 6-month-old P301S mice for 3 months would influence phospho-tau (p-tau) accumulation, tau insolubility, and neurodegeneration. RESULTS: Treatment with HJ8.5 at 50 mg/kg showed a very strong decrease in detergent-insoluble tau. Importantly, HJ8.5 significantly reduced the loss of cortical and hippocampal tissue volumes compared to control treated mice. HJ8.5 treatment reduced hippocampal CA1 cellular layer staining with the p-tau antibody AT8 and thio-S-positive tau aggregates in piriform cortex and amygdala. Moreover, mice treated with HJ8.5 at 50 mg/kg showed a decrease in motor/sensorimotor deficits compared to vehicle-treated mice. Some effects of HJ8.5, including reduction in brain atrophy, and p-tau immunostaining were also seen with a dose of 10 mg kg(-1) week(-1). In BV2-microglial cells, we observed significantly higher uptake of P301S tau aggregates in the presence of HJ8.5. HJ8.5 treatment also resulted in a large dose-dependent increase of tau in the plasma. INTERPRETATION: Our results indicate that systemically administered anti-tau antibody HJ8.5 significantly decreases insoluble tau, decreases brain atrophy, and improves motor/sensorimotor function in a mouse model of tauopathy. These data further support the idea that anti-tau antibodies should be further assessed as a potential treatment for tauopathies.

Previously we proposed that transmission of the hedgehog signal across the plasma membrane by Smoothened is triggered by its interaction with cholesterol (Luchetti et al., 2016). But how is cholesterol, an abundant lipid, regulated tightly enough to control a signaling system that can cause birth defects and cancer? Using toxin-based sensors that distinguish between distinct pools of cholesterol, we find that Smoothened activation and Hedgehog signaling are driven by a biochemically-defined, small fraction of membrane cholesterol, termed accessible cholesterol. Increasing cholesterol accessibility by depletion of sphingomyelin, which sequesters cholesterol in complexes, amplifies Hedgehog signaling. Hedgehog ligands increase cholesterol accessibility in the membrane of the primary cilium by inactivating the transporter-like protein Patched 1. Trapping this accessible cholesterol blocks Hedgehog signal transmission across the membrane. Our work shows that the organization of cholesterol in the ciliary membrane can be modified by extracellular ligands to control the activity of cilia-localized signaling proteins.

Lysosomes coordinate cellular metabolism and growth upon sensing of essential nutrients, including cholesterol. Through bioinformatic analysis of lysosomal proteomes, we identified lysosomal cholesterol signaling (LYCHOS, previously annotated as G protein-coupled receptor 155), a multidomain transmembrane protein that enables cholesterol-dependent activation of the master growth regulator, the protein kinase mechanistic target of rapamycin complex 1 (mTORC1). Cholesterol bound to the amino-terminal permease-like region of LYCHOS, and mutating this site impaired mTORC1 activation. At high cholesterol concentrations, LYCHOS bound to the GATOR1 complex, a guanosine triphosphatase (GTPase)-activating protein for the Rag GTPases, through a conserved cytoplasm-facing loop. By sequestering GATOR1, LYCHOS promotes cholesterol- and Rag-dependent recruitment of mTORC1 to lysosomes. Thus, LYCHOS functions in a lysosomal pathway for cholesterol sensing and couples cholesterol concentrations to mTORC1-dependent anabolic signaling.

< 0.001). These results suggest that 25% nitrous oxide has comparable efficacy to 50% nitrous oxide in improving TRMD but with a markedly lower rate of adverse effects.

Apolipoprotein E (apoE) is the strongest known genetic risk factor for late onset Alzheimer's disease (AD). It influences amyloid-β (Aβ) clearance and aggregation, which likely contributes in large part to its role in AD pathogenesis. We recently found that HJ6.3, a monoclonal antibody against apoE, significantly reduced Aβ plaque load when given to APPswe/PS1ΔE9 (APP/PS1) mice starting before the onset of plaque deposition. To determine whether the anti-apoE antibody HJ6.3 affects Aβ plaques, neuronal network function, and behavior in APP/PS1 mice after plaque onset, we administered HJ6.3 (10 mg/kg/week) or PBS intraperitoneally to 7-month-old APP/PS1 mice for 21 weeks. HJ6.3 mildly improved spatial learning performance in the water maze, restored resting-state functional connectivity, and modestly reduced brain Aβ plaque load. There was no effect of HJ6.3 on total plasma cholesterol or cerebral amyloid angiopathy. To investigate the underlying mechanisms of anti-apoE immunotherapy, HJ6.3 was applied to the brain cortical surface and amyloid deposition was followed over 2 weeks using in vivo imaging. Acute exposure to HJ6.3 affected the course of amyloid deposition in that it prevented the formation of new amyloid deposits, limited their growth, and was associated with occasional clearance of plaques, a process likely associated with direct binding to amyloid aggregates. Topical application of HJ6.3 for only 14 d also decreased the density of amyloid plaques assessed postmortem. Collectively, these studies suggest that anti-apoE antibodies have therapeutic potential when given before or after the onset of Aβ pathology.

Metabotropic glutamate receptor 5 (mGluR5) is widely expressed throughout the CNS and participates in regulating neuronal function and synaptic transmission. Recent work in the striatum led to the groundbreaking discovery that intracellular mGluR5 activation drives unique signaling pathways, including upregulation of ERK1/2, Elk-1 (Jong et al., 2009) and Arc (Kumar et al., 2012). To determine whether mGluR5 signals from intracellular membranes of other cell types, such as excitatory pyramidal neurons in the hippocampus, we used dissociated rat CA1 hippocampal cultures and slice preparations to localize and characterize endogenous receptors. As in the striatum, CA1 neurons exhibited an abundance of mGluR5 both on the cell surface and intracellular membranes, including the endoplasmic reticulum and the nucleus where it colocalized with the sodium-dependent excitatory amino acid transporter, EAAT3. Inhibition of EAAT3 or sodium-free buffer conditions prevented accumulations of radiolabeled agonist. Using a pharmacological approach to isolate different pools of mGluR5, both intracellular and cell surface receptors induced oscillatory Ca(2+) responses in dissociated CA1 neurons; however, only intracellular mGluR5 activation triggered sustained high amplitude Ca(2+) rises in dendrites. Consistent with the notion that mGluR5 can signal from intracellular membranes, uncaging glutamate on a CA1 dendrite led to a local Ca(2+) rise, even in the presence of ionotropic and cell surface metabotropic receptor inhibitors. Finally, activation of intracellular mGluR5 alone mediated both electrically induced and chemically induced long-term depression, but not long-term potentiation, in acute hippocampal slices. These data suggest a physiologically relevant and important role for intracellular mGluR5 in hippocampal synaptic plasticity.

Neurosteroids are endogenous modulators of neuronal excitability and nervous system development and are being developed as anesthetic agents and treatments for psychiatric diseases. While gamma amino-butyric acid Type A (GABAA) receptors are the primary molecular targets of neurosteroid action, the structural details of neurosteroid binding to these proteins remain ill defined. We synthesized neurosteroid analogue photolabeling reagents in which the photolabeling groups were placed at three positions around the neurosteroid ring structure, enabling identification of binding sites and mapping of neurosteroid orientation within these sites. Using middle-down mass spectrometry (MS), we identified three clusters of photolabeled residues representing three distinct neurosteroid binding sites in the human α1β3 GABAA receptor. Novel intrasubunit binding sites were identified within the transmembrane helical bundles of both the α1 (labeled residues α1-N408, Y415) and β3 (labeled residue β3-Y442) subunits, adjacent to the extracellular domains (ECDs). An intersubunit site (labeled residues β3-L294 and G308) in the interface between the β3(+) and α1(-) subunits of the GABAA receptor pentamer was also identified. Computational docking studies of neurosteroid to the three sites predicted critical residues contributing to neurosteroid interaction with the GABAA receptors. Electrophysiological studies of receptors with mutations based on these predictions (α1-V227W, N408A/Y411F, and Q242L) indicate that both the α1 intrasubunit and β3-α1 intersubunit sites are critical for neurosteroid action.

Allopregnanolone is synthesized in the central nervous system either de novo from cholesterol or from steroid hormone precursors like progesterone and pregnenolone. Over the past 30 years, direct and rapid, non-genomic actions of allopregnanolone and its derivatives via GABAA receptors have been demonstrated. Changes in brain levels of allopregnanolone during pregnancy and in the postpartum period, or during exposure to protracted stress appear to play a crucial role in the pathophysiology of mood disorders. The discovery that allopregnanolone at low (nanomolar) concentrations elicits marked anxiolytic, anti-stress and antidepressant effects by facilitating allosterically the action of GABA at extrasynaptic GABAA receptors has provided new perspectives for the discovery of novel drugs useful for the treatment of mood disorders. These findings have led to the seminal clinical studies that recently demonstrated that treatment with allopregnanolone (i.e., brexanolone) can dramatically and rapidly improve the symptoms of postpartum depression in many patients.

The major cholesterol metabolite in brain, 24(S)-hydroxycholesterol (24S-HC), serves as a vehicle for cholesterol removal. Its effects on neuronal function, however, have only recently begun to be investigated. Here, we review that nascent work. Our own studies have demonstrated that 24S-HC has potent positive modulatory effects on N-methyl-d-aspartate (NMDA) receptor (NMDAR) function. This could have implications not only for brain plasticity but also for pathological NMDAR overuse. Other work has demonstrated effects of 24S-HC on neuronal survival and as a possible biomarker of neurodegenerative disease. Depending on circumstances, both upregulation/mimicry of 24S-HC signaling and down-regulation/antagonism may have therapeutic potential. We are interested in the possibility that synthetic analogues of 24S-HC with positive effects at NMDARs may hold neurotherapeutic promise, given the role of NMDA receptor hypofunction in certain neuropsychiatric disorders.

Mutations in DNA methyltransferase 3A (DNMT3A) have been detected in autism and related disorders, but how these mutations disrupt nervous system function is unknown. Here, we define the effects of DNMT3A mutations associated with neurodevelopmental disease. We show that diverse mutations affect different aspects of protein activity but lead to shared deficiencies in neuronal DNA methylation. Heterozygous DNMT3A knockout mice mimicking DNMT3A disruption in disease display growth and behavioral alterations consistent with human phenotypes. Strikingly, in these mice, we detect global disruption of neuron-enriched non-CG DNA methylation, a binding site for the Rett syndrome protein MeCP2. Loss of this methylation leads to enhancer and gene dysregulation that overlaps with models of Rett syndrome and autism. These findings define the effects of DNMT3A haploinsufficiency in the brain and uncover disruption of the non-CG methylation pathway as a convergence point across neurodevelopmental disorders.

Nicotinamide adenine dinucleotide (NAD + ) is a critical coenzyme for cellular energy metabolism. The aim of the present study was to determine the importance of brown and white adipose tissue (BAT and WAT) NAD + metabolism in regulating whole-body thermogenesis and energy metabolism. Accordingly, we generated and analyzed adipocyte-specific nicotinamide phosphoribosyltransferase ( Nampt ) knockout (ANKO) and brown adipocyte-specific Nampt knockout (BANKO) mice because NAMPT is the rate-limiting NAD + biosynthetic enzyme. We found ANKO mice, which lack NAMPT in both BAT and WAT, had impaired gene programs involved in thermogenesis and mitochondrial function in BAT and a blunted thermogenic (rectal temperature, BAT temperature, and whole-body oxygen consumption) response to acute cold exposure, prolonged fasting, and administration of β-adrenergic agonists (norepinephrine and CL-316243). In addition, the absence of NAMPT in WAT markedly reduced adrenergic-mediated lipolytic activity, likely through inactivation of the NAD + –SIRT1–caveolin-1 axis, which limits an important fuel source fatty acid for BAT thermogenesis. These metabolic abnormalities were rescued by treatment with nicotinamide mononucleotide (NMN), which bypasses the block in NAD + synthesis induced by NAMPT deficiency. Although BANKO mice, which lack NAMPT in BAT only, had BAT cellular alterations similar to the ANKO mice, BANKO mice had normal thermogenic and lipolytic responses. We also found NAMPT expression in supraclavicular adipose tissue (where human BAT is localized) obtained from human subjects increased during cold exposure, suggesting our finding in rodents could apply to people. These results demonstrate that adipose NAMPT-mediated NAD + biosynthesis is essential for regulating adaptive thermogenesis, lipolysis, and whole-body energy metabolism.

The relative contributions of glycolysis and oxidative phosphorylation to neuronal presynaptic energy demands are unclear. In rat hippocampal neurons, ATP production by either glycolysis or oxidative phosphorylation alone sustained basal evoked synaptic transmission for up to 20 min. However, combined inhibition of both ATP sources abolished evoked transmission. Neither action potential propagation failure nor depressed Ca 2+ influx explained loss of evoked synaptic transmission. Rather, inhibition of ATP synthesis caused massive spontaneous vesicle exocytosis, followed by arrested endocytosis, accounting for the disappearance of evoked postsynaptic currents. In contrast to its weak effects on basal transmission, inhibition of oxidative phosphorylation alone depressed recovery from vesicle depletion. Local astrocytic lactate shuttling was not required. Instead, either ambient monocarboxylates or neuronal glycolysis was sufficient to supply requisite substrate. In summary, basal transmission can be sustained by glycolysis, but strong presynaptic demands are met preferentially by oxidative phosphorylation, which can be maintained by bulk but not local monocarboxylates or by neuronal glycolysis. SIGNIFICANCE STATEMENT Neuronal energy levels are critical for proper CNS function, but the relative roles for the two main sources of ATP production, glycolysis and oxidative phosphorylation, in fueling presynaptic function in unclear. Either glycolysis or oxidative phosphorylation can fuel low-frequency synaptic function and inhibiting both underlies loss of synaptic transmission via massive vesicle release and subsequent failure to endocytose lost vesicles. Oxidative phosphorylation, fueled by either glycolysis or endogenously released monocarboxylates, can fuel more metabolically demanding tasks such as vesicle recovery after depletion. Our work demonstrates the flexible nature of fueling presynaptic function to maintain synaptic function.

A wide variety of animal models have been used to study glaucoma. Although these models provide valuable information about the disease, there is still no ideal model for studying glaucoma due to its complex pathogenesis. Animal models for glaucoma are pivotal for clarifying glaucoma etiology and for developing novel therapeutic strategies to halt disease progression. In this review paper, we summarize some of the major findings obtained in various glaucoma models and examine the strengths and limitations of these models.

Neuroinflammation, a major hallmark of Alzheimer's disease and several other neurological and psychiatric disorders, is often associated with dysregulated cholesterol metabolism. Relative to homeostatic microglia, activated microglia express higher levels of Ch25h, an enzyme that hydroxylates cholesterol to produce 25-hydroxycholesterol (25HC). 25HC is an oxysterol with interesting immune roles stemming from its ability to regulate cholesterol metabolism. Since astrocytes synthesize cholesterol in the brain and transport it to other cells via ApoE-containing lipoproteins, we hypothesized that secreted 25HC from microglia may influence lipid metabolism as well as extracellular ApoE derived from astrocytes. Here, we show that astrocytes take up externally added 25HC and respond with altered lipid metabolism. Extracellular levels of ApoE lipoprotein particles increased after treatment of astrocytes with 25HC without an increase in Apoe mRNA expression. In mouse astrocytes-expressing human ApoE3 or ApoE4, 25HC promoted extracellular ApoE3 better than ApoE4. Increased extracellular ApoE was due to elevated efflux from increased Abca1 expression via LXRs as well as decreased lipoprotein reuptake from suppressed Ldlr expression via inhibition of SREBP. 25HC also suppressed expression of Srebf2, but not Srebf1, leading to reduced cholesterol synthesis in astrocytes without affecting fatty acid levels. We further show that 25HC promoted the activity of sterol-o-acyl transferase that led to a doubling of the amount of cholesteryl esters and their concomitant storage in lipid droplets. Our results demonstrate an important role for 25HC in regulating astrocyte lipid metabolism.

N-methyl-D-aspartate receptors (NMDARs), a major subtype of glutamate receptors mediating excitatory transmission throughout the central nervous system (CNS), play critical roles in governing brain function and cognition. Because NMDAR dysfunction contributes to the etiology of neurological and psychiatric disorders including stroke and schizophrenia, NMDAR modulators are potential drug candidates. Our group recently demonstrated that the major brain cholesterol metabolite, 24S-hydroxycholesterol (24S-HC), positively modulates NMDARs when exogenously administered. Here, we studied whether endogenous 24S-HC regulates NMDAR activity in hippocampal slices. In CYP46A1(-/-) (knockout; KO) slices where endogenous 24S-HC is greatly reduced, NMDAR tone, measured as NMDAR-to-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) excitatory postsynaptic current (EPSC) ratio, was reduced. This difference translated into more NMDAR-driven spiking in wild-type (WT) slices compared with KO slices. Application of SGE-301, a 24S-HC analog, had comparable potentiating effects on NMDAR EPSCs in both WT and KO slices, suggesting that endogenous 24S-HC does not saturate its NMDAR modulatory site in ex vivo slices. KO slices did not differ from WT slices in either spontaneous neurotransmission or in neuronal intrinsic excitability, and exhibited LTP indistinguishable from WT slices. However, KO slices exhibited higher resistance to persistent NMDAR-dependent depression of synaptic transmission induced by oxygen-glucose deprivation (OGD), an effect restored by SGE-301. Together, our results suggest that loss of positive NMDAR tone does not elicit compensatory changes in excitability or transmission, but it protects transmission against NMDAR-mediated dysfunction. We expect that manipulating this endogenous NMDAR modulator may offer new treatment strategies for neuropsychiatric dysfunction.

Smoothened (SMO) transduces the Hedgehog (Hh) signal across the plasma membrane in response to accessible cholesterol. Cholesterol binds SMO at two sites: one in the extracellular cysteine-rich domain (CRD) and a second in the transmembrane domain (TMD). How these two sterol-binding sites mediate SMO activation in response to the ligand Sonic Hedgehog (SHH) remains unknown. We find that mutations in the CRD (but not the TMD) reduce the fold increase in SMO activity triggered by SHH. SHH also promotes the photocrosslinking of a sterol analog to the CRD in intact cells. In contrast, sterol binding to the TMD site boosts SMO activity regardless of SHH exposure. Mutational and computational analyses show that these sites are in allosteric communication despite being 45 angstroms apart. Hence, sterols function as both SHH-regulated orthosteric ligands at the CRD and allosteric ligands at the TMD to regulate SMO activity and Hh signaling.

Abstract Cognitive dysfunction is one of the most concerning outcomes in global population aging. However, the mechanisms by which cognitive functions are impaired during aging remain elusive. It has been established that NAD + levels are reduced in multiple tissues and organs, including the brain. We found that NAD + levels declined in the hippocampus of mice during the course of aging, and whereas we observed minimal age-related effects on spatial learning/memory capabilities in old mice, we discovered that they developed cognitive hypersensitivity in response to aversive stimulation during contextual fear conditioning tests. This cognitive hypersensitivity appears to be associated with alterations in emotionality (fear/anxiety) and sensory processing (shock sensitivity), rather than reflect genuine conditioning/retention effects, during aging. Supplementation of nicotinamide mononucleotide (NMN) improved the sensory processing aspect of the hypersensitivity and possibly other related behaviors. Specific knockdown of nicotinamide phosphoribosyltransferase ( Nampt ) in the CA1 region, but not in the dentate gyrus, recapitulates this cognitive hypersensitivity observed in old mice. We identified calcium/calmodulin-dependent serine protein kinase ( Cask ) as a potential downstream effector in response to age-associated NAD + reduction in the hippocampus. Cask expression is responsive to NAD + changes and also reduced in the hippocampus during aging. Short-term NMN supplementation can enhance Cask expression in the hippocampus of old mice. Its promoter activity is regulated in a Sirt1-dependent manner. Taken together, NAD + reduction in the CA1 region contributes to development of age-associated cognitive dysfunction, aspects of which may be prevented or treated by enhancing NAD + availability through supplementation of NAD + intermediates, such as NMN.