Matthew L. MacDonald

Antipsychotic drugs elevate mRNA levels of presynaptic proteins in the frontal cortex of the rat

BACKGROUND Molecular adaptations are believed to contribute to the mechanism of action of antipsychotic drugs (APDs). We attempted to establish common gene regulation patterns induced by chronic treatment with APDs. METHODS Gene expression analysis was performed with the Affymetrix U34A array in the frontal cortex (FC) and the striatum of rats chronically treated with two concentrations of either clozapine or haloperidol. Key data were verified with real-time quantitative polymerase chain reaction. RESULTS Many genes in the FC affected by APD-treatment contribute to similar functions. mRNAs coding for synaptic vesicle docking- and microtubule-associated proteins were upregulated; mRNAs for serine-threonine protein phosphatases were downregulated, whereas the serine-threonine kinases protein kinase A, protein kinase C, and calcium/calmodulin kinase II alpha and IV were upregulated, indicating increased potential for protein phosphorylation. In the striatum, altered gene expression was less focused on genes of particular function or location, and the high concentration of haloperidol had a different gene expression profile than any of the other APD treatments. CONCLUSION We found an increase in the transcription of genes coding for proteins involved in synaptic plasticity and synaptic activity in the FC. We furthermore found that the gene expression profile of APDs is different between FC and striatum.

The glutamate hypothesis of schizophrenia: evidence from human brain tissue studies.

A number of studies have indicated that antagonists of the N‐methyl‐d‐aspartate subtypes of glutamate receptors can cause schizophrenia‐like symptoms in healthy individuals and exacerbate symptoms in individuals with schizophrenia. These findings have led to the glutamate hypothesis of schizophrenia. Here we review the evidence for this hypothesis in postmortem studies of brain tissue from individuals affected by schizophrenia, summarizing studies of glutamate neuron morphology, of expression of glutamate receptors and transporters, and of the synthesizing and metabolizing enzymes for glutamate and its co‐agonists. We found consistent evidence of morphological alterations of dendrites of glutamatergic neurons in the cerebral cortex of subjects with schizophrenia and of reduced levels of the axon bouton marker synaptophysin. There were no consistent alterations of mRNA expression of glutamate receptors, although there has been limited study of the corresponding proteins. Studies of the glutamate metabolic pathway have been limited, although there is some evidence that excitatory amino acid transporter‐2, glutamine synthetase, and glutaminase have altered expression in schizophrenia. Future studies would benefit from additional direct examination of glutamatergic proteins. Further advances, such as selective testing of synaptic microdomains, cortical layers, and neuronal subtypes, may also be required to elucidate the nature of glutamate signaling impairments in schizophrenia.

The Post-Synaptic Density of Human Postmortem Brain Tissues: An Experimental Study Paradigm for Neuropsychiatric Illnesses

Recent molecular genetics studies have suggested various trans-synaptic processes for pathophysiologic mechanisms of neuropsychiatric illnesses. Examination of pre- and post-synaptic scaffolds in the brains of patients would greatly aid further investigation, yet such an approach in human postmortem tissue has yet to be tested. We have examined three methods using density gradient based purification of synaptosomes followed by detergent extraction (Method 1) and the pH based differential extraction of synaptic membranes (Methods 2 and 3). All three methods separated fractions from human postmortem brains that were highly enriched in typical PSD proteins, almost to the exclusion of pre-synaptic proteins. We examined these fractions using electron microscopy (EM) and verified the integrity of the synaptic membrane and PSD fractions derived from human postmortem brain tissues. We analyzed protein composition of the PSD fractions using two dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) and observed known PSD proteins by mass spectrometry. Immunoprecipitation and immunoblot studies revealed that expected protein-protein interactions and certain posttranscriptional modulations were maintained in PSD fractions. Our results demonstrate that PSD fractions can be isolated from human postmortem brain tissues with a reasonable degree of integrity. This approach may foster novel postmortem brain research paradigms in which the stoichiometry and protein composition of specific microdomains are examined.

Isolation rearing and hyperlocomotion are associated with reduced immediate early gene expression levels in the medial prefrontal cortex

Environmental deprivation contributes in important ways to the development of a wide range of psychiatric disorders. Isolation rearing of rodents, a model for environmental deprivation in humans, consistently produces hyperlocomotion, which provides a measurable parameter to study the underlying mechanisms of early adverse psychosocial stressors. Male Sprague-Dawley rat pups were separated from dams at postnatal (PN) day 20 and reared either in groups of three or in isolation. On PN 38, locomotion was assessed in the open field. On PN 46, rats were killed and gene expression patterns examined in the medial prefrontal cortex (mPFC). Isolation-reared rats displayed increased locomotor activity and decreased resting time in the open field. Specific gene expression patterns in the mPFC were associated with both isolation rearing and hyperlocomotive behavior in the open field. Genes involved in these expression patterns included immediate early genes (IEGs) and genes that regulate cell differentiation and apoptosis. The study of these genes could provide important insights into how abnormal early psychosocial events affect brain function and behavior.

Loss of Microtubule-Associated Protein 2 Immunoreactivity Linked to Dendritic Spine Loss in Schizophrenia

BACKGROUND Microtubule-associated protein 2 (MAP2) is a neuronal protein that plays a role in maintaining dendritic structure through its interaction with microtubules. In schizophrenia (Sz), numerous studies have revealed that the typically robust immunoreactivity (IR) of MAP2 is significantly reduced across several cortical regions. The relationship between MAP2-IR reduction and lower dendritic spine density, which is frequently reported in Sz, has not been explored in previous studies, and MAP2-IR loss has not been investigated in the primary auditory cortex (Brodmann area 41), a site of conserved pathology in Sz. METHODS Using quantitative spinning disk confocal microscopy in two cohorts of subjects with Sz and matched control subjects (Sz subjects, n = 20; control subjects, n = 20), we measured MAP2-IR and dendritic spine density and spine number in deep layer 3 of BA41. RESULTS Subjects with Sz exhibited a significant reduction in MAP2-IR. The reductions in MAP2-IR were not associated with neuron loss, loss of MAP2 protein, clinical confounders, or technical factors. Dendritic spine density and number also were reduced in Sz and correlated with MAP2-IR. In 12 (60%) subjects with Sz, MAP2-IR values were lower than the lowest values in control subjects; only in this group were spine density and number significantly reduced. CONCLUSIONS These findings demonstrate that MAP2-IR loss is closely linked to dendritic spine pathology in Sz. Because MAP2 shares substantial sequence, regulatory, and functional homology with MAP tau, the wealth of knowledge regarding tau biology and the rapidly expanding field of tau therapeutics provide resources for identifying how MAP2 is altered in Sz and possible leads to novel therapeutics.

Human olfactory epithelial cells generated in vitro express diverse neuronal characteristics

The olfactory epithelium constitutes the sole source of regenerating neural cells that can be obtained from a living human. As such, primary cultures derived from human olfactory epithelial biopsies can be utilized to study neurobiological characteristics of individuals under different conditions and disease states. Here, using such human cultures, we report in vitro generation of cells that exhibit a complex neuronal phenotype, encompassing receptors and signaling pathways pertinent to both olfaction and other aspects of CNS function. Using in situ hybridization, we demonstrate for the first time the native expression of olfactory receptors in cultured cells derived from human olfactory epithelial tissue. We further establish the presence and function of olfactory transduction molecules in these cells using immunocytochemistry, calcium imaging and molecular methods. Western blot analysis revealed the expression of neurotransmitter receptors for dopamine (D2R), 5-HT (5HT2C) and NMDA subtypes 1 and 2A/2B. Stimulation with dopamine or 5-HT enhanced receptor G protein activation in a subtype specific manner, based on 35S-guanosine triphosphate incorporation assay. Functional characteristics of the cultured cells are demonstrated through enhanced tyrosine phosphorylation of NMDAR 2A/2B and recruitment of signaling partners in response to NMDA stimulation. The array of neuronal characteristics observed here establishes that proliferating cells derived from the human olfactory epithelium differentiate in vitro to express functional and molecular attributes of mature olfactory neurons. These cultured neural cells exhibit neurotransmitter pathways important in a number of neuropsychiatric disorders. Their ready availability from living humans thus provides a new tool to link functional and molecular features of neural cells with clinical characteristics of individual living patients.

Altered Glutamate Protein Co-Expression Network Topology Linked to Spine Loss in the Auditory Cortex of Schizophrenia

BACKGROUND Impaired glutamatergic signaling is believed to underlie auditory cortex pyramidal neuron dendritic spine loss and auditory symptoms in schizophrenia. Many schizophrenia risk loci converge on the synaptic glutamate signaling network. We therefore hypothesized that alterations in glutamate signaling protein expression and co-expression network features are present in schizophrenia. METHODS Gray matter homogenates were prepared from auditory cortex gray matter of 22 schizophrenia and 23 matched control subjects, a subset of whom had been previously assessed for dendritic spine density. One hundred fifty-five selected synaptic proteins were quantified by targeted mass spectrometry. Protein co-expression networks were constructed using weighted gene co-expression network analysis. RESULTS Proteins with evidence for altered expression in schizophrenia were significantly enriched for glutamate signaling pathway proteins (GRIA4, GRIA3, ATP1A3, and GNAQ). Synaptic protein co-expression was significantly decreased in schizophrenia with the exception of a small group of postsynaptic density proteins, whose co-expression increased and inversely correlated with spine density in schizophrenia subjects. CONCLUSIONS We observed alterations in the expression of glutamate signaling pathway proteins. Among these, the novel observation of reduced ATP1A3 expression is supported by strong genetic evidence indicating it may contribute to psychosis and cognitive impairment phenotypes. The observations of altered protein network topology further highlight the complexity of glutamate signaling network pathology in schizophrenia and provide a framework for evaluating future experiments to model the contribution of genetic risk to disease pathology.

Large-scale generation of human iPSC-derived neural stem cells/early neural progenitor cells and their neuronal differentiation

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented opportunity to perform high-throughput screening of novel drugs for neurological and neurodegenerative diseases. Such screenings require a robust and scalable method for generating large numbers of mature, differentiated neuronal cells. Currently available methods based on differentiation of embryoid bodies (EBs) or directed differentiation of adherent culture systems are either expensive or are not scalable. We developed a protocol for large-scale generation of neuronal stem cells (NSCs)/early neural progenitor cells (eNPCs) and their differentiation into neurons. Our scalable protocol allows robust and cost-effective generation of NSCs/eNPCs from iPSCs. Following culture in neurobasal medium supplemented with B27 and BDNF, NSCs/eNPCs differentiate predominantly into vesicular glutamate transporter 1 (VGLUT1) positive neurons. Targeted mass spectrometry analysis demonstrates that iPSC-derived neurons express ligand-gated channels and other synaptic proteins and whole-cell patch-clamp experiments indicate that these channels are functional. The robust and cost-effective differentiation protocol described here for large-scale generation of NSCs/eNPCs and their differentiation into neurons paves the way for automated high-throughput screening of drugs for neurological and neurodegenerative diseases.

Biochemical Fractionation and Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry for Targeted and Microdomain-Specific Protein Quantification in Human Postmortem Brain Tissue

Synaptic architecture and its adaptive changes require numerous molecular events that are both highly ordered and complex. A majority of neuropsychiatric illnesses are complex trait disorders, in which multiple etiologic factors converge at the synapse via many signaling pathways. Investigating the protein composition of synaptic microdomains from human patient brain tissues will yield valuable insights into the interactions of risk genes in many disorders. These types of studies in postmortem tissues have been limited by the lack of proper study paradigms. Thus, it is necessary not only to develop strategies to quantify protein and post-translational modifications at the synapse, but also to rigorously validate them for use in postmortem human brain tissues. In this study we describe the development of a liquid chromatography-selected reaction monitoring method, using a stable isotope-labeled neuronal proteome standard prepared from the brain tissue of a stable isotope-labeled mouse, for the multiplexed quantification of target synaptic proteins in mammalian samples. Additionally, we report the use of this method to validate a biochemical approach for the preparation of synaptic microdomain enrichments from human postmortem prefrontal cortex. Our data demonstrate that a targeted mass spectrometry approach with a true neuronal proteome standard facilitates accurate and precise quantification of over 100 synaptic proteins in mammalian samples, with the potential to quantify over 1000 proteins. Using this method, we found that protein enrichments in subcellular fractions prepared from human postmortem brain tissue were strikingly similar to those prepared from fresh mouse brain tissue. These findings demonstrate that biochemical fractionation methods paired with targeted proteomic strategies can be used in human brain tissues, with important implications for the study of neuropsychiatric disease.

Selective Loss of Smaller Spines in Schizophrenia

OBJECTIVE Decreased density of dendritic spines in adult schizophrenia subjects has been hypothesized to result from increased pruning of excess synapses in adolescence. In vivo imaging studies have confirmed that synaptic pruning is largely driven by the loss of large or mature synapses. Thus, increased pruning throughout adolescence would likely result in a deficit of large spines in adulthood. Here, the authors examined the density and volume of dendritic spines in deep layer 3 of the auditory cortex of 20 schizophrenia and 20 matched comparison subjects as well as aberrant voltage-gated calcium channel subunit protein expression linked to spine loss. METHOD Primary auditory cortex deep layer 3 spine density and volume was assessed in 20 pairs of schizophrenia and matched comparison subjects in an initial and replication cohort (12 and eight pairs) by immunohistochemistry-confocal microscopy. Targeted mass spectrometry was used to quantify postsynaptic density and voltage-gated calcium channel protein expression. The effect of increased voltage-gated calcium channel subunit protein expression on spine density and volume was assessed in primary rat neuronal culture. RESULTS Only the smallest spines are lost in deep layer 3 of the primary auditory cortex in subjects with schizophrenia, while larger spines are retained. Levels of the tryptic peptide ALFDFLK, found in the schizophrenia risk gene CACNB4, are inversely correlated with the density of smaller, but not larger, spines in schizophrenia subjects. Consistent with this observation, CACNB4 overexpression resulted in a lower density of smaller spines in primary neuronal cultures. CONCLUSIONS These findings require a rethinking of the overpruning hypothesis, demonstrate a link between small spine loss and a schizophrenia risk gene, and should spur more in-depth investigations of the mechanisms that govern new or small spine generation and stabilization under normal conditions as well as how this process is impaired in schizophrenia.