BS3 Chemical Crosslinking Assay: Evaluating the Effect of Chronic Stress on Cell Surface GABA<sub>A</sub> Receptor Presentation in the Rodent Brain

Anxiety is a state of emotion that variably affects animal behaviors, including cognitive functions. Behavioral signs of anxiety are observed across the ...

Campbell Family Mental Health Research Institute

University of Toronto

Alcoholism is a psychiatric condition that develops through neuroadaptations in response to neuronal stresses caused by chronic ethanol intake. Neurons can adapt to ethanol-induced metabolic changes by activating cellular protective mechanisms, including autophagy. Here we show that expression of Ulk1, a gene critical to the regulation of autophagy, was affected in the prefrontal cortex (PFC) of mice following chronic intermittent ethanol (CIE) exposure. Consequently, overall levels of Ulk1 activity in the PFC were downregulated, leading to accumulation of p62, a protein that serves as a target for autophagic degradation. In addition, Ulk1-null mice demonstrated decline in the exploratory activity, deficits in the ability to recognize novel objects following CIE exposure, and reduced rate of voluntary ethanol drinking. The data suggest the neuroprotective role for Ulk1-mediated autophagy in the suppression of neuropsychiatric manifestation during ethanol exposure.

Ulk1 protects against ethanol-induced neuronal stress and cognition-related behavioral deficits.

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder that, while prevalent, has a stagnant track record for advances in treatment. The limited availability of animal models with appropriate face and predictive validities has hampered progress in developing novel neurobiological hypotheses and testing new therapeutic options for this condition. Here, we report that mice deficient in , a gene specifically expressed in the nervous system with documented functions in neurodevelopment, show hyperactivity and impulsivity phenotypes, which are ameliorated by administering methylphenidate (MPH) or guanfacine (GFC), two pharmacological agents used for ADHD treatment. -knockout (KO) mice show reduced expression of tyrosine hydroxylase in the midbrain and the brain stem and have reduced levels of dopamine, norepinephrine, or their metabolites in both the nucleus accumbens and the prefrontal cortex. These neurochemical changes in -KO mice were normalized by MPH or GFC. We propose that -KO mice can be used as a model to evaluate the role of altered neurodevelopment in the manifestation of ADHD-like behavioral phenotypes, as well as to investigate the neurobiological mechanisms of existing and new pharmacotherapeutic agents for ADHD.

Methylphenidate and Guanfacine Ameliorate ADHD-Like Phenotypes in -Deficient Mice.

Autophagy plays an essential role in intracellular degradation and maintenance of cellular homeostasis in all cells, including neurons. Although a recent study reported a copy number variation of Ulk2, a gene essential for initiating autophagy, associated with a case of schizophrenia (SZ), it remains to be studied whether Ulk2 dysfunction could underlie the pathophysiology of the disease. Here we show that Ulk2 heterozygous (Ulk2+/-) mice have upregulated expression of sequestosome-1/p62, an autophagy-associated stress response protein, predominantly in pyramidal neurons of the prefrontal cortex (PFC), and exhibit behavioral deficits associated with the PFC functions, including attenuated sensorimotor gating and impaired cognition. Ulk2+/- neurons showed imbalanced excitatory-inhibitory neurotransmission, due in part to selective down-modulation of gamma-aminobutyric acid (GABA)A receptor surface expression in pyramidal neurons. Genetically reducing p62 gene dosage or suppressing p62 protein levels with an autophagy-inducing agent restored the GABAA receptor surface expression and rescued the behavioral deficits in Ulk2+/- mice. Moreover, expressing a short peptide that specifically interferes with the interaction of p62 and GABAA receptor-associated protein, a protein that regulates endocytic trafficking of GABAA receptors, also restored the GABAA receptor surface expression and rescued the behavioral deficits in Ulk2+/- mice. Thus, the current study reveals a novel mechanism linking deregulated autophagy to functional disturbances of the nervous system relevant to SZ, through regulation of GABAA receptor surface presentation in pyramidal neurons.

Ulk2 controls cortical excitatory-inhibitory balance via autophagic regulation of p62 and GABAA receptor trafficking in pyramidal neurons.

Individuals with chromosome 22q11.2 deletions are at increased risk of developing psychiatric conditions, most notably, schizophrenia (SZ). Recently, clinical studies have also implicated these recurrent 22q11.2 deletions with the risk of early-onset Parkinson's disease (PD). Thus far, the multiple mouse models generated for 22q11.2 deletions have been studied primarily in the context of congenital cardiac, neurodevelopmental, and psychotic disorders. One of these is the /+ model, in which SZ-associated and developmental abnormalities have been reported. We present the first evidence that the mouse model for the 22q11.2 deletion exhibits motor coordination deficits and molecular signatures (that is, elevated α-synuclein expression) relevant to PD. Reducing the α-synuclein gene dosage in /+ mice ameliorated the motor deficits. Thus, this model of the 22q11.2 deletion shows signatures of both SZ and PD at the molecular and behavioral levels. In addition, both SZ-associated and PD-relevant deficits in the model were ameliorated by treatment with a rapamycin analog, CCI-779. We now posit the utility of 22q11.2 deletion mouse models in investigating the mechanisms of SZ- and PD-associated manifestations that could shed light on possible common pathways of these neuropsychiatric disorders.

A mouse model of 22q11.2 deletions: Molecular and behavioral signatures of Parkinson's disease and schizophrenia.

Aging is associated with reduced brain volume, altered neural activity, and neuronal atrophy in cortical-like structures, comprising the frontal cortex and hippocampus, together contributing to cognitive impairments. Therapeutic efforts aimed at reversing these deficits have focused on excitatory or neurotrophic mechanisms, although recent findings show that reduced dendritic inhibition mediated by α5-subunit containing GABA-A receptors (α5-GABAA-Rs) occurs during aging and contributes to cognitive impairment. Here, we aimed to confirm the beneficial effect on working memory of augmenting α5-GABAA-R activity in old mice and tested its potential at reversing age-related neuronal atrophy. We show that GL-II-73, a novel ligand with positive allosteric modulatory activity at α5-GABAA-R (α5-PAM), increases dendritic branching complexity and spine numbers of cortical neurons in vitro. Using old mice, we confirm that α5-PAM reverses age-related working memory deficits and show that chronic treatment (3 months) significantly reverses age-related dendritic shrinkage and spine loss in frontal cortex and hippocampus. A subsequent 1-week treatment cessation (separate cohort) resulted in loss of efficacy on working memory but maintained morphological neurotrophic effects. Together, the results demonstrate the beneficial effect on working memory and neurotrophic efficacy of augmenting α5-GABAA-R function in old mice, suggesting symptomatic and disease-modifying potential in age-related brain disorders.

Reversal of Age-Related Neuronal Atrophy by α5-GABAA Receptor Positive Allosteric Modulation.

Reduced brain-derived neurotrophic factor (BDNF) and gamma-aminobutyric acid (GABA) neurotransmission co-occur in brain conditions (depression, schizophrenia and age-related disorders) and are associated with symptomatology. Rodent studies show they are causally linked, suggesting the presence of biological pathways mediating this link. Here we first show that reduced BDNF and GABA also co-occur with attenuated autophagy in human depression. Using mice, we then show that reducing Bdnf levels (Bdnf) leads to upregulated sequestosome-1/p62, a key autophagy-associated adaptor protein, whose levels are inversely correlated with autophagic activity. Reduced Bdnf levels also caused reduced surface presentation of α5 subunit-containing GABA receptor (α5-GABAR) in prefrontal cortex (PFC) pyramidal neurons. Reducing p62 gene dosage restored α5-GABAR surface expression and rescued PFC-relevant behavioral deficits of Bdnf mice, including cognitive inflexibility and reduced sensorimotor gating. Increasing p62 levels was sufficient to recreate the molecular and behavioral profiles of Bdnf mice. Collectively, the data reveal a novel mechanism by which deficient BDNF leads to targeted reduced GABAergic signaling through autophagic dysregulation of p62, potentially underlying cognitive impairment across brain conditions.

BDNF controls GABAR trafficking and related cognitive processes via autophagic regulation of p62.

Neural circuit functions critically depend on homeostatic regulation and quality control of neuronal proteins and organelles. Emerging evidence shows that autophagy, cellular clearance machinery, selectively degrades or controls homeostasis of both pre- and post-synaptic components (e.g. synaptic proteins, organelles, neurotransmitters, and their receptors), thereby regulating synaptic remodeling, neurotransmission, and neuroplasticity. Along with its well-known role in supporting neuronal cell viability and neurodevelopment, autophagy is now implicated in a wide range of neuronal physiology throughout neuronal lifetime, including higher-order brain functions such as information processing, memory encoding, or cognitive functions. Here, we review recent literature on the roles of neuronal autophagy in homeostatic maintenance of synaptic functions and discuss how disruptions in these processes may contribute to the pathophysiology of neurodevelopmental and psychiatric disorders.

Autophagy in neuronal physiology and disease.

Major depressive disorder (MDD) is a brain disorder often characterized by recurrent episode and remission phases. The molecular correlates of MDD have been investigated in case-control comparisons, but the biological alterations associated with illness trait (regardless of clinical phase) or current state (symptomatic and remitted phases) remain largely unknown, limiting targeted drug discovery. To characterize MDD trait- and state-dependent changes, in single or recurrent depressive episode or remission, we generated transcriptomic profiles of subgenual anterior cingulate cortex of postmortem subjects in first MDD episode (n = 20), in remission after a single episode (n = 15), in recurrent episode (n = 20), in remission after recurring episodes (n = 15) and control subject (n = 20). We analyzed the data at the gene, biological pathway, and cell-specific molecular levels, investigated putative causal events and therapeutic leads. MDD-trait was associated with genes involved in inflammation, immune activation, and reduced bioenergetics (q < 0.05) whereas MDD-states were associated with altered neuronal structure and reduced neurotransmission (q < 0.05). Cell-level deconvolution of transcriptomic data showed significant change in density of GABAergic interneurons positive for corticotropin-releasing hormone, somatostatin, or vasoactive-intestinal peptide (p < 3 × 10). A probabilistic Bayesian-network approach showed causal roles of immune-system-activation (q < 8.67 × 10), cytokine-response (q < 4.79 × 10) and oxidative-stress (q < 2.05 × 10) across MDD-phases. Gene-sets associated with these putative causal changes show inverse associations with the transcriptomic effects of dopaminergic and monoaminergic ligands. The study provides first insights into distinct cellular and molecular pathologies associated with trait- and state-MDD, on plasticity mechanisms linking the two pathologies, and on a method of drug discovery focused on putative disease-causing pathways.

Molecular characterization of depression trait and state.

Metabolic dysfunction is thought to contribute to the severity of psychiatric disorders; however, it has been unclear whether current high–simple sugar diets contribute to pathogenesis of these diseases. Here, we demonstrate that a high-sucrose diet during adolescence induces psychosis-related behavioral endophenotypes, including hyperactivity, poor working memory, impaired sensory gating, and disrupted interneuron function in mice deficient for glyoxalase-1 (GLO1), an enzyme involved in detoxification of sucrose metabolites. Furthermore, the high-sucrose diet induced microcapillary impairments and reduced brain glucose uptake in brains of -deficient mice. Aspirin protected against this angiopathy, enhancing brain glucose uptake and preventing abnormal behavioral phenotypes. Similar vascular damage to our model mice was found in the brains of randomly collected schizophrenia and bipolar disorder patients, suggesting that psychiatric disorders are associated with angiopathy in the brain caused by various environmental stresses, including metabolic stress.

High-sucrose diets contribute to brain angiopathy with impaired glucose uptake and psychosis-related higher brain dysfunctions in mice.

Reduced somatostatin (SST) and dysfunction of SST-positive (SST) neurons are hallmarks of neurological disorders and associated with mood disturbances, but the molecular origin of SST neuron vulnerability is unknown. Using chronic psychosocial stress as a paradigm to induce elevated behavioral emotionality in rodents, we report a selective vulnerability of SST neurons through exacerbated unfolded protein response (UPR) of the endoplasmic reticulum (ER), or ER stress, in the prefrontal cortex. We next show that genetically suppressing ER stress in SST neurons, but not in pyramidal neurons, normalized behavioral emotionality induced by psychosocial stress. In search for intrinsic factors mediating SST neuron vulnerability, we found that the forced expression of the SST precursor protein (preproSST) in SST neurons, mimicking psychosocial stress-induced early proteomic changes, induces ER stress, whereas mature SST or processing-incompetent preproSST does not. Biochemical analyses further show that psychosocial stress induces SST protein aggregation under elevated ER stress conditions. These results demonstrate that SST processing in the ER is a SST neuron-intrinsic vulnerability factor under conditions of sustained or over-activated UPR, hence negatively impacting SST neuron functions. Combined with observations in major medical illness, such as diabetes, where excess ER processing of preproinsulin similarly causes ER stress and β cell dysfunction, this suggests a universal mechanism for proteinopathy that is induced by excess processing of native endogenous proteins, playing critical pathophysiological roles that extend to neuropsychiatric disorders.

Molecular origin of somatostatin-positive neuron vulnerability.

Sensorimotor information processing underlies normal cognitive and behavioral traits and has classically been evaluated through prepulse inhibition (PPI) of a startle reflex. PPI is a behavioral dimension deregulated in several neurological and psychiatric disorders, yet the mechanisms underlying the cross-diagnostic nature of PPI deficits across these conditions remain to be understood. To identify circuitry mechanisms for PPI, we performed circuitry recording over the prefrontal cortex and striatum, two brain regions previously implicated in PPI, using wild-type (WT) mice compared to Disc1-locus-impairment (LI) mice, a model representing neuropsychiatric conditions. We demonstrated that the corticostriatal projection regulates neurophysiological responses during the PPI testing in WT, whereas these circuitry responses were disrupted in Disc1-LI mice. Because our biochemical analyses revealed attenuated brain-derived neurotrophic factor (Bdnf) transport along the corticostriatal circuit in Disc1-LI mice, we investigated the potential role of Bdnf in this circuitry for regulation of PPI. Virus-mediated delivery of Bdnf into the striatum rescued PPI deficits in Disc1-LI mice. Pharmacologically augmenting Bdnf transport by chronic lithium administration, partly via phosphorylation of Huntingtin (Htt) serine-421 and its integration into the motor machinery, restored striatal Bdnf levels and rescued PPI deficits in Disc1-LI mice. Furthermore, reducing the cortical Bdnf expression negated this rescuing effect of lithium, confirming the key role of Bdnf in lithium-mediated PPI rescuing. Collectively, the data suggest that striatal Bdnf supply, collaboratively regulated by Htt and Disc1 along the corticostriatal circuit, is involved in sensorimotor gating, highlighting the utility of dimensional approach in investigating pathophysiological mechanisms across neuropsychiatric disorders.

Toshifumi Tomoda

Centre for Addiction and Mental Health (CAMH)

Campbell Family Mental Health Research Institute

BS3 Chemical Crosslinking Assay: Evaluating the Effect of Chronic Stress on Cell Surface GABA_A Receptor Presentation in the Rodent Brain

Toshifumi Tomoda

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Centre for Addiction and Mental Health (CAMH)

Campbell Family Mental Health Research Institute

BS3 Chemical Crosslinking Assay: Evaluating the Effect of Chronic Stress on Cell Surface GABAA Receptor Presentation in the Rodent Brain

Centre for Addiction and Mental Health (CAMH)

BS3 Chemical Crosslinking Assay: Evaluating the Effect of Chronic Stress on Cell Surface GABA_A Receptor Presentation in the Rodent Brain