Koko Ishizuka, M.D., Ph.D. - US grants

DESCRIPTION (provided by applicant): Bipolar disorder (BP) is among the most important public health problems in the world and is one of the top ten leading causes of lifelong disability Recent large-scale collaborative sample collections along with rapid advances in genome technology are poised to provide statistical evidence of association and/or linkage, but mechanistic understanding of the disease remains at an initial stage. One major limitation that has blocked the progress, although mental disorders such as BP and schizophrenia affect the brain, is the difficulty in accessing neuronal cells from patients. To overcome this dilemma, we have optimized a protocol to enrich olfactory immature neurons from nasal biopsied samples from humans. We previously utilized this methodology to study molecular and cellular profiles of patients with schizophrenia (SZ) and normal controls. Here in this proposal, we plan to use the olfactory epithelium immature neurons (OE neurons) from subjects with psychotic BP, which is familiarly associated with SZ and shares many endophenotypic abnormalities, such as greater severity of cognitive deficits and impaired pre-pulse inhibition. Thus, we hypothesize that such shared abnormalities may stem from molecular and cellular deficits associated with neurodevelopment. To address this hypothesis, we will newly recruit 30 subjects with psychotic and non-psychotic BP, and enrich OE neurons (Aim 1). By using these cells, we will determine development-associated deficits, with an emphasis on Wnt pathway and developmental switch of phospho-DISC1, in psychotic BP. The data will be compared with those from OE neurons from patients with schizophrenia, non-psychotic BP, and normal controls (Aim 2). Then, we will identify gene expression profiles related to Wnt signaling/cilia formation pathways in OE neurons from patients with psychotic BP, in comparison with those from patients with schizophrenia, non-psychotic BP, and controls (Aim 3). This study has the potential to provide important insight into the molecular and cellular signature of psychotic BP associated with neurodevelopment. We expect that some of the major changes may be shared with those in schizophrenia, which contribute to the further study of common susceptibility mechanisms for these disorders. PUBLIC HEALTH RELEVANCE: Based on promising preliminary data, we perform gene expression and functional studies in olfactory neurons from bipolar disorder to address its molecular mechanisms.

DESCRIPTION (provided by applicant): The diagnostic boundaries defined by current diagnostic systems (e.g., DSM) are now being challenged by recent advances in the genetic architecture underlying psychiatric disorders. Meanwhile, NIMH has launched the Research Domain Criteria project (RDoC) to develop, for research purposes, new ways of classifying psychopathology based on dimensions of observable behavior and neurobiological measures beyond the current diagnostic systems. The DISC1 gene was originally discovered as the sole disrupted transcript with an open reading frame, at the breakpoint of an inherited chromosomal translocation in a Scottish pedigree: it segregates with a variety of major mental illnesses, including schizophrenia (SZ), bipolar disorder (BP), and major depression. Nonetheless, genome wide association studies have failed to detect associations between DISC1 locus and SZ (or other DSM-categorized diseases thus far). In contrast, the data from association studies of DISC1 with anatomical, physiological, and behavioral traits, which commonly underlie the pathology of major mental illnesses, have been promising. Thus, we hypothesize that DISC1 is a promising target to address mechanisms underlying mental illnesses across diagnostic categories in the RDoC framework: DISC1 may be a good probe that mediates translation from the discoveries in basic genetics, neuroscience, and behavioral science into clinical application. Based on our preliminary data, we further hypothesize that a decrease in the level of phosphorylation at serine-713 of human DISC1 (pS713-DISC1) underlies delayed neural differentiation, which disturbs neural circuitry formation in the brain development and, in turn, interferes with the acquisition of working memory. We will study relatively stable outpatients with SZ and BP, as well as well-matched healthy controls. Within the RDoC framework, our construct of interest is working memory (cognitive domain), whereas the independent variable is pS713-DISC1 (molecule). Our dependent variables include neuronal fate (cells), cortical surface area, thickness, and volume (circuit), and working memory (behavior). Johns Hopkins Schizophrenia Center has established an infrastructure of translational research in which we conduct clinical/neuropsychological assessment, brain imaging, and multiple tissue biopsies for molecular and cellular study simultaneously from each study participant. This infrastructure allows us to perform experiments in which molecular, anatomical, and behavioral data will be obtained from the same individuals. By utilizing this potential strength, we will address how behavior and neuroanatomical abnormalities relevant to psychotic disorders (SZ and BP currently categorized by DSM) are quantitatively associated with a specific molecular signature (phosphorylation of DISC1 in this study).

Abstract In many areas of medicine, pathophysiological studies of biospecimens from living patients have facilitated our understanding of disease mechanisms. However, due to the difficulty of brain biopsies, such strategies have not been developed for Alzheimer?s disease (AD). Thus, there is a great need for novel methodologies to collect neurons from living patients in order to capture pathological changes relevant to AD. The olfactory neuroepithelium has received great interest as a surrogate tissue to study brain disorders. Multiple studies have shown the neuronal validity of olfactory neurons/neuronal cells at the molecular level. As the olfactory neuroepithelium includes neural stem cells/neuroprogenitor cells, the tissue can quickly regenerate following a biopsy, which allows us to repeat biopsies over time without impairing olfactory function. However, the classic nasal biopsy platform is limited by its invasiveness and the insufficient purity of neurons in the biopsied tissue. To overcome these limitations, we recently developed a new platform: a soft nasal brush swab followed by a single-cell analysis specific for neurons. This new platform is now even easier and even less invasive than a blood draw, and neural purity is guaranteed at a single-cell resolution. Olfactory dysfunction is an early symptom preceding robust memory deficits in AD; A? lesions and tau pathology occur extensively in the olfactory system, including the olfactory neuroepithelium. Therefore, we hypothesized that phosphorylated tau protein at threonine 181(pT181-tau) detected in olfactory neurons through single-cell Western blotting is increased in AD patients, compared to subjects with mild cognitive impairment (MCI) and controls, and additionally that such pT181-tau levels detected in olfactory neurons are associated with the pT181-tau levels detected in cerebrospinal fluid (CSF) and with neurocognitive function. Indeed, we found that pT181-tau levels in olfactory neurons obtained from AD patients were significantly higher than those from cognitively normal controls in our pilot study. Encouraged by this promising data, we will study pT181-tau levels in olfactory neurons at the single-cell resolution in 30 patients with AD, 30 subjects with MCI, and 30 cognitively normal controls (Aim 1); We will compare pT181-tau levels in olfactory neurons detected at the single-cell level with those detected in CSF from the same individuals (Aim 2); We will determine whether pT181-tau levels in olfactory neurons detected at the single-cell level are associated with neuropsychological function (Aim 3). Through this proof-of-concept study, we hope to establish a novel, high-throughput, and non- invasive platform (nasal brush swab followed by single-cell Western blotting) to study the pathophysiology of AD in neurons obtained from living patients at the single-cell level.