Lecture 10 - Modelling Schizophrenia

PDGFRa

glial progenitor cell marker, had IPSPs from SCZ kids and wanted to see if when transplanted in mice via transplantation it recreated SCZ

what is the rationale behind astroglial cell changes causing SCZ?

studies have highlighted the frequent dysregulation of glia-selective genes both astrocytic and oligodendrocytic in SCZ vs controls looking at parents and siblings to understand genetics there, patients with SCZ typically are characterized by lower levels of white matter and hypomyelination so reduced myelin in certain areas of the brain caused by oligodendrocytes (form myelin, found in white matter so changes in these cells indicates issues with myelination_

clinical background

pathological and neuroimaging studies have highlighted deficiencies in oligodendroglial density and myelin structure in affected patients, recent studies have also emphasized role of oligodendrocytes in metabolic support of neurons suggesting myelin-independent mechanisms where oligodendrocyti dysfunction might yield neuronal pathology, however despite genetic, cellular, pathological and imaging studies have correlated glial and myelin pathology with schizophrenia, many researchers have simply assumed that clinical hypomyelination among schizophrenics is secondary to neuronal pathology, as a result the contribution of glial dysfunction in schizophrenia has not been well studied

what technique does this paper use?

makes us of chimeric mice and tech for reprogramming fibroblasts back into glial progenitor cells that can become either oligodendrocytes or astroglia, prepared hGPCs) from iPSCs derived from fibroblasts taken from either juvenile onset patients with SCZ or normal controls, assessed the differential gene expression of SCZ hGPCs relative to controls, transplanted the cells into immunodeficient neonatal mice to produce patient specific human glial chimeric mice, the glial chimeric mice were then analyzed regarding the effects of SCZ derivation on astrocytic and oligodendrocytic differentiation in vivo as well as behavioral phenotype and whether its correlated to disease-associated gene expression

overview of paper rationale

SCZ is a heritable psychiatric disorder involving impairments in cognition, perception and motivation that usually manifest late in adolescence or early in adulthood, the pathogenic mechanisms underlying SCZ are unknown but researchers have repeatedly noted pathological features involving excessive loss of grey matter, also commonly observe reduced numbers of synaptic structures on neurons

what area of SCZ research still needs to be investigated?

even though there are pharmalogical treatments for psychotic symptoms of schizophrenia, there is no clear mechanistic understanding or effective therapies to prevent or treat cognitive impairments and deficit symptoms of SCZ which are earliest and most common features of the disorder, need to find biological processes that underlie such disorders to help us understand the disease process as well

dopamine involvement in SCZ

dopaminergic overactivity has been a predominant pathophysiologic hypothesis of SCZ causing the psychotic symptoms, evidence to support this is the effects of agents that stimulate dopamine outflow like psychostimulant amphetamine, psychostimulants can produce a paranoid psychosis in healthy ppl and cause symptomatic worsening in approx 1/3 of patients with SCZ, supports hypothesis that at least a subgroup of SCZ is associated with increased synaptic dopamine concentrations

iPSCs and lentiviruses

iPSCs can be used to develop structures that look like organoids via lentiviruses, once we have fibroblasts we can use lentiviruses carrying their polycistronic plasmid vector to deliver more than 1 gene to the cell at a time, so we can deliver all 4 yamanaka factors in one gene and infect every single fibroblast to make them pluripotent

Yamanaka factors

Myc, Oct4, Sox2 and Klf4

how do we test for stemness?

all lines were initially characterized and validated as pluripotent using global RNA sequencing to assess pluripotent gene expression as well as immunostaining for Oct4, Nanog and SSEA4 + Sox2 and TRA 1-60 which all mark de-differentiated nano-potent stem cells, they can also do a teratoma assay

hochest stain

confirms every cell has pluripotent marker, stains everything with DNA

teratoma assay

test for stemness by injecting iPSC colony in SCID mouse, a teratoma is a tumor with stem cells, so basically what we do is take a group of iPSCs in culture, put it into back of SCID mouse and see if cells divide and proliferate to make a tumor, should be able to see all cell types present if its a teratoma (mesodermal, ectodermal and endodermal cells), start with iPSCs and see if they can be pushed into something similar to glial progenitor cells

STR DNA profiling

use a lentivirus to re-program fully differentiated cells back to their pluripotent origins so they have to sequence and do RNA profiling, use STR DNA (2-5 bp DNA sequences that are repeated several times in succession), the chromosome 7 one is very polymorphic and often associated with SCZ, to prove hypothesis have to convert SCZ and control iPSCs into glial progenitor cells via injecting yamanaka factors into them,

hypomyelination shiverer mice in the experiment

transplant either the SCZ or control GPC cell line into hypomyelinated shiverer mice (shake cause of uncoordinated muscle movement), want to examine myelination in a hypomyelinated mice, then it will go on to form oligodendrocytes and want to know if the SCZ ones are able to form myelin, if we wanna see if there is an issue with myelination wanna see if the cells can myelinate hypomyelinated neurons, control cells migrate in the mice to many areas of the brain (lots of red indicating lots of movement in the mice), in the SCZ line marked with human nuclei see less migration/survival of these cells, less oligodendrocytes present in SCZ glial progenitor cell line

MBP

mature oligodendrocyte marker, see less of these cells in SCZ line compared to the control line so less green staining

SCZ GPCs

the ones that entered the gray matter might have differentiated into astrocytes in that environment or they might have impairment in lineage progression that prevents their astrocytic differentiation (not only oligodendrocytes but astrocytes might be affect in SCZ too) cause astrocyte differentiation is also lower/deficient in SCZ, use hGFAP (astrocyte marker) expression comparing between controls and SCZ line and see much less present in SCZ compared to controls (less morphology and amount present)

RNA sequencing analysis

RNA sequence analysis that SCZ hGPCs have different gene expression compared to controls, these changes are reflected in down regulation of channel activity glutamate receptor activity anterograde trans-synaptic signaling, chemical synaptic transmission modulation of synaptic transmission, chemical synaptic transmission modulation of synaptic transmission, central nervous system development, oligodendrocyte differentiation, glial cell differentiation, myelination, ensheathment of neurons galactolipid biosynthesis process

NXNPH1, NLGN3 and LINGO1

synaptic genes whose dysregulation has been previously linked to both SCZ and autism spectrum disorder and found to be down regulated via RNA-seq, leads to less functional connectivity, synaptic integrity and myelination

NgR

binding of OMgp, MAG and Nogo to this receptor leads to high affinity to inhibit neurite growth and myelination, it also interacts with LINGO-1 to transduce inhibitory signals, helps with signaling and neuronal development, interactions between oligodendrocytes and diseases found to affect signaling in SCZ, MS, depression, etc

experimental procedure of RNA seq

synaptic associated genes (NRXN1, NLGN1, DSCAML1 and SLITRKs 2-5) were sharply downregulated in hGPCs derived from 3 of the 4 patients, when the RNA seq data shows a trend, use quantitative real-time PCR to compare SCZ and control data, and what the transcript levels for each being compared to, what is the reference or control gene (ex: GAPDH is a reference gene), qPCR is able to show synapse associated genes are downregulated hGPCs cause its targeted, qualitative and a precise method

does the glial cell differences in SCZ line affect behavioral phenotype?

the abberrant filtration of hGPCs and their resulting astroglia into the developing cortex might influence information processing within the cortex when mature, some past studies report influence of astrocytic networks on synaptic efficacy and plasticity and the differential competence of human glia in this respect

what do behavioral tests of SCZ examine?

using mice allows investigation of genetic, neurochemical and behavioral factors, behavioral paradigms mimic schizophrenia symptoms in humans, core domains studied include cognitive dysfunction, social behavior deficits (dont socialize as much) and sensorimotor gating impairments (problems with filtering out auditory information)

novel object recognition

rodents explore two identical objects, after a delay, one object is replaced with a novel one, exploration time is recorded, for SCZ it assesses working and recognition memory deficits, linked to HPC and prefrontal cortex dysfunction, an example would be comparing time spent exploring novel object in control vs SCZ model rodents, the control animal would be expected to spend more time with the novel object compared to SCZ animal

social interaction test

measures time spent interacting with another rodent in an open field or a three chambered box, this is relevant to SCZ cause it models social withdrawal observed in SCZ, a core negative symptom, its linked to altered oxytocin and dopamine pathways, comparing social interaction time across experimental groups

Y maze alternation test

examines PFC, tests if animal remembers which arm of the maze it explored vs the new one its going to, rodents have to choose between three arms of a maze, spontaneous alternation is used as a measure of working memory, relevant to SCZ cause assesses deficits in executive function and decision making linked to PFC dysfunction,

open field test

measures total distance traveled and exploratory behavior in an open area, if the animals show positive thigmotaxis they will display anxiety and hug the wall rather than explore the open field, this is relevant to SCZ cause hyperlocomotion models psychotic-like behavior due to increased dopamine + anxiety means you move less, linked to dopamine hyperactivity,

anxiety/fear models

rodents learn to associate a neutral stimulus (tone) with an aversive event (shock), freezing behavior is measured, assesses associative learning and fear processing deficits, linked to amygdala and hippocampal dysfunction, an alternative test is the elevated plus maze

pre-pulse inhibition of the acoustic startle response

this is a widely used measure in mouse models of SCZ, PPI refers to the reduction in the startle response when a weaker non startling stimulus (prepulse) precedes a sudden, loud noise (pulse), this process reflects sensorimotor gating (the brain’s ability to filter out irrelevant stimuli), deficits in PPI are commonly observed in patients with SCZ indicating impaired sensorimotor gating,

pre-pulse inhibition trial

the lighter sound before the big one preps mice so it wont get startled, in SCZ they wont learn this cause they dont have sensorimotor gating so large startle reaction even for the lighter sound, they think both sounds are equally scary, in the model inhibit the PPI with NMDA receptor antagonists like MK-801 and dopamine agonists

PPI in schizophrenia

in mouse models of SCZ, PPI deficits can be induced through genetic modifications like mutations in SCZ related genes like DISC1 or NRG1, pharmacological interventions like administration of dopamine agonists or NMDA receptor antagonists like ketamine or environmental factors like prenatal stress or early-life adversity, restoration of PPI through antipsychotic drugs like dopamine antagonists or glutamatergic modulators further validates this model as a tool for studying SCZ’ PPI testing in mice is a valuable translational approach for investigating sensorimotor gating deficits and evaluating new therapeutic strategies for SCZ

conclusions

glial distribution and SCZ types of hGPCs were found to alter the myelination and behavior of the engrafted chimeric mice, altered astrocytic development in the SCZ type engrafted mice suggesting that maturation of astrocytes may be a result of lower levels of migration and glial distribution/myelination, there is yet another role for glial cells in behaviours or outcomes that we would normally find associated with the human brain, not neuronal genes only