Protein insolubility has been found to occur in a subset of patients with schizophrenia.
Dissolving the pathogenesis of schizophrenia.
Schizophrenia is a heterogeneous disorder. Subtyping patients based on the presence of specific pathological processes may increase our understanding of illness pathophysiology. Abnormal protein confirmation and solubility may play a role in many disorders, including schizophrenia. There is evidence that the protein products of certain genes linked to schizophrenia are prone to insolubility.1 There is also evidence for abnormal ubiquitin insolubility and proteins in brain tissue from patients with schizophrenia, which may modulate protein insolubility and be a marker for insoluble protein aggregates.2
Nucifora and colleagues3 tested the hypothesis that protein insolubility and ubiquitination are present in some brains from patients with schizophrenia and investigated the potential biological relevance of these insoluble proteins. They first performed a pilot study using tissue from the prefrontal cortex of autopsy brains from the Harvard Brain Tissue Resource Center. Next, they performed a full-scale study using tissue from the prefrontal cortex and superior temporal gyrus from autopsy brains from the University of Pittsburgh Brain Bank. They also performed a replication study using tissue from the prefrontal cortex from autopsy brains from the University of Texas Southwestern Medical Center.
Homogenates from brain tissue were prepared to obtain the insoluble fraction. The insoluble fractions were then subjected to electrophoresis (SDS-PAGE), followed by Coomassie/silver staining to quantify total homogenate protein and protein insolubility. Western blot analysis was used to investigate ubiquitin. Insoluble protein pellets were also solubilized and analyzed by liquid chromatography-tandem mass spectrometry. Demographic and biochemical characteristics of brain samples from patients with schizophrenia were compared with controls subjects in each of the three brain bank cohorts. Hierarchical clustering analysis was performed to identify brains with and without protein insolubility and ubiquitin reactivity. Proteins identified by mass spectrometry were analyzed with both gene set and cell type enrichment, to identify dysregulated pathways and cell types contributing to or resulting from protein insolubility.
The pilot study consisted of brain tissue from five patients with schizophrenia and four controls. The full-scale study included brain tissue from 19 patients with schizophrenia and 19 controls. In both the pilot and full-scale studies, a subset of patients with schizophrenia had significantly more protein insolubility and ubiquitination than both other patients and controls. Based on cluster analysis, 10 of 19 brains of patients with schizophrenia had high protein insolubility and ubiquitination. Importantly, the total amount of starting protein was not significantly different between subject groups, suggesting a shift in the amount of protein from the soluble to insoluble fraction in that subset of patients. In the full-scale study, brain pH was significantly lower in patients (versus controls), but there were no differences in age, sex, race, post-mortem interval, and RNA integrity. Brain pH was also lower, and anticonvulsant exposure was higher in patients with schizophrenia with (versus without) protein abnormalities.
In the replication sample, which consisted of brain tissue from 18 patients with schizophrenia and 18 controls, cluster analysis identified eight brains of patients with schizophrenia and high protein insolubility and ubiquitination compared to other subjects. There were no differences in age, sex, race, post-mortem interval, brain pH, and RNA integrity between subject groups.
In order to investigate potential effects of antipsychotic treatment of protein abnormalities, the authors treated rats for 4.5 months with haloperidol or risperidone. Using the same protocol, they found no evidence of medication-induced changes in protein insolubility or ubiquitination, suggesting the observed associations in humans are independent of the effects of antipsychotic medication.
Among proteins significantly different between patients with high insolubility and ubiquitination, gene enrichment analysis identified axon target recognition as the biological process of highest significance. Pathway analysis identified neurological disease, cell assembly and organization, and nervous system development and function as the top pathways that differed between subject groups. The authors also found that insoluble proteins may be enriched in specific neuronal subtypes, including GABAergic neurons, cortical and subcortical projection neurons, and granule neurons.
The authors concluded that they identified a subset of brains for patients with schizophrenia with increased protein insolubility and ubiquitination, and in greater abundance, in cortical tissue. The insoluble proteins are disproportionally reflective of pathways related to nervous system development and axon target recognition. The authors noted the consistency of findings across samples from three different brain banks, and across different cortical regions.
The Bottom Line
Findings support the hypothesis that protein insolubility occurs in a subset of patients with schizophrenia, and that the insolubility may be related to disease pathogenesis.
Dr Miller is Associate Professor of Psychiatry, Department of Psychiatry and Health Behavior, Augusta University, Augusta, Georgia. He is the Schizophrenia Section Editor for Psychiatric Times.
The author reports that he receives research support from Augusta University, the National Institute of Mental Health, the Brain and Behavior Research Foundation, and the Stanley Medical Research Institute.
1. Nucifora LG, Wu YC, Lee BJ, et al. A mutation in NPAS3 that segregates with schizophrenia in a small family leads to protein aggregation. Mol Neuropsychiatry. 2016;2:133–144
2. Rubio MD, Wood K, Haroutunian V, et al: Dysfunction of the ubiquitin proteasome and ubiquitin-like systems in schizophrenia. Neuropsychopharmacology. 2013;38:1910–1920.
3. Nucifora LG, MacDonald ML, Lee BJ, et al. Increased Protein Insolubility in Brains From a Subset of Patients With Schizophrenia. Am J Psychiatry. 2019; doi: 10.1176/appi.ajp.2019.18070864