Neural Progenitor Pool Research Articles

Longitudinal tracking of hemocyte populations in vivo indicates lineage relationships and supports neural progenitor identity in adult neurogenesis

Adult neurogenesis, which takes place in both vertebrate and invertebrate species, is the process by which new neurons are born and integrated into existing functional neural circuits, long after embryonic development. Most studies in mammals suggest that self-renewing stem cells are the source of the new neurons, although the extent of self-renewal is a matter of debate. In contrast, research in the crayfish Procambarus clarkii has demonstrated that the neural progenitors producing adult-born neurons are capable of both self-renewing and consuming (non-self-renewing) divisions. However, self-renewing divisions are relatively rare, and therefore the production of adult-born neurons depends heavily on progenitors that are not replenishing themselves. Because the small pool of neural progenitors in the neurogenic niche is never exhausted throughout the long lives of these animals, we hypothesized that there must also be an extrinsic source of these cells. It was subsequently demonstrated that the neural progenitors originate in hemocytes (blood cells) produced by the immune system that travel in the circulation before ultimately integrating into niches where the neural lineage begins. The current study examines the developmental lineage of the three hemocyte types — hyaline (HC), semigranular (SGC) and granular (GC) cells — with the goal of understanding the origins of the progenitor cells that produce adult-born neurons. Longstanding qualitative metrics for hemocyte classification were validated quantitatively. Then, in a longitudinal study, proliferation markers were used to label the hemocytes in vivo, followed by sampling the circulating hemocyte population over the course of two months. Hemolymph samples were taken at intervals to track the frequencies of the different hemocyte types. These data reveal sequential peaks in the relative frequencies of HCs, SGCs and GCs, which were identified using qualitative and quantitative measures. These findings suggest that the three hemocyte types comprise a single cellular lineage that occurs in the circulation, with each type as a sequential progressive stage in hemocyte maturation beginning with HCs and ending with GCs. When combined with previously published data, this timeline provides additional evidence that HCs serve as the primary neural progenitor during adult neurogenesis in P. clarkii.

In a zebrafish biomedical model of human Allan-Herndon-Dudley syndrome impaired MTH signaling leads to decreased neural cell diversity.

Maternally derived thyroid hormone (T3) is a fundamental factor for vertebrate neurodevelopment. In humans, mutations on the thyroid hormones (TH) exclusive transporter monocarboxylic acid transporter 8 (MCT8) lead to the Allan-Herndon-Dudley syndrome (AHDS). Patients with AHDS present severe underdevelopment of the central nervous system, with profound cognitive and locomotor consequences. Functional impairment of zebrafish T3 exclusive membrane transporter Mct8 phenocopies many symptoms observed in patients with AHDS, thus providing an outstanding animal model to study this human condition. In addition, it was previously shown in the zebrafish mct8 KD model that maternal T3 (MTH) acts as an integrator of different key developmental pathways during zebrafish development. Using a zebrafish Mct8 knockdown model, with consequent inhibition of maternal thyroid hormones (MTH) uptake to the target cells, we analyzed genes modulated by MTH by qPCR in a temporal series from the start of segmentation through hatching. Survival (TUNEL) and proliferation (PH3) of neural progenitor cells (dla, her2) were determined, and the cellular distribution of neural MTH-target genes in the spinal cord during development was characterized. In addition, in-vivo live imaging was performed to access NOTCH overexpression action on cell division in this AHDS model. We determined the developmental time window when MTH is required for appropriate CNS development in the zebrafish; MTH is not involved in neuroectoderm specification but is fundamental in the early stages of neurogenesis by promoting the maintenance of specific neural progenitor populations. MTH signaling is required for developing different neural cell types and maintaining spinal cord cytoarchitecture, and modulation of NOTCH signaling in a non-autonomous cell manner is involved in this process. The findings show that MTH allows the enrichment of neural progenitor pools, regulating the cell diversity output observed by the end of embryogenesis and that Mct8 impairment restricts CNS development. This work contributes to the understanding of the cellular mechanisms underlying human AHDS.

Long-term Culture of Cerebral Organoid Reveals Disruption of Glial Cell Differentiation and Glutamate Excitotoxicity in Multiple Sclerosis. (P2-3.002)

<h3>Objective:</h3> Analyze differentiation and maturation of glial cells and neurons in long-term cultured cerebral organoids derived from induced pluripotent stem cells of patients with multiple sclerosis (PwMS). <h3>Background:</h3> MS is an auto-immune disease characterized by inflammation, demyelination and neural degeneration. It has been shown that environmental and genetic factors contribute to the development of MS, however, the etiology of this condition is unknown. Our previous research showed that organoids from PwMS display an exhaustion of the neural progenitor pool and an increase in neurogenesis. Here, we studied differentiation of neurons into glutamate and GABAergic neurons as glutamate excitotoxicity has been proposed to lead to inflammation and neuronal injury in MS. We also investigated glial cell differentiation and maturation in astrocytes and oligodendrocytes to determine their role in MS pathogenesis. <h3>Design/Methods:</h3> We analyzed neural cell differentiation in neuronal and glial lineages along with myelination capacity by immunofluorescence in MS cerebral organoids compared to healthy control organoids, after 120 days in vitro. <h3>Results:</h3> <i>A</i> strong decrease of Olig2 expression in MS organoids was detected highlighting a lower number of oligodendrocyte lineage cells. Moreover, no colocalization of Olig2 and APC was observed in MS, while multiple double positive cells were detected in control organoids, showing a defect of oligodendrocytes maturation. In parallel, GFAP staining showed a decrease of GFAP expression in MS organoids compared to control. We then analyzed GABAergic and glutamatergic differentiation. A strong increase of glutamatergic marker vGlut1 and a decrease of GABAergic marker GAD67 was detected at d120 in MS organoids compared to control signifying an imbalance of excitatory and inhibitory neurons. <h3>Conclusions:</h3> Mature cerebral organoids developed from PwMS represent an innovative tool to study MS and allowed us to detect a defect of astrocyte and oligodendrocyte differentiation and maturation as well as an imbalance of GABA/Glutamate release in MS organoids <b>Disclosure:</b> Mr. Mehta has nothing to disclose. Ms. McDermott has nothing to disclose. Nicolas Daviaud has nothing to disclose. Dr. Sadiq has nothing to disclose.

PQBP1 regulates striatum development through balancing striatal progenitor proliferation and differentiation

The balance between cell proliferation and differentiation is essential for maintaining the neural progenitor pool and brain development. Although the mechanisms underlying cell proliferation and differentiation at the transcriptional level have been studied intensively, post-transcriptional regulation of cell proliferation and differentiation remains largely unclear. Here, we show that deletion of the alternative splicing regulator PQBP1 in striatal progenitors results in defective striatal development due to impaired neurogenesis of spiny projection neurons (SPNs). Pqbp1-deficient striatal progenitors exhibit declined proliferation and increased differentiation, resulting in a reduced striatal progenitor pool. We further reveal that PQBP1 associates with components in splicing machinery. The alternative splicing profiles identify that PQBP1 promotes the exon 9 inclusion of Numb, a variant that mediates progenitor proliferation. These findings identify PQBP1 as a regulator in balancing striatal progenitor proliferation and differentiation and provide alternative insights into the pathogenic mechanisms underlying Renpenning syndrome.

An Optimized Workflow to Generate and Characterize iPSC-Derived Motor Neuron (MN) Spheroids.

A multitude of in vitro models based on induced pluripotent stem cell (iPSC)-derived motor neurons (MNs) have been developed to investigate the underlying causes of selective MN degeneration in motor neuron diseases (MNDs). For instance, spheroids are simple 3D models that have the potential to be generated in large numbers that can be used across different assays. In this study, we generated MN spheroids and developed a workflow to analyze them. To start, the morphological profiling of the spheroids was achieved by developing a pipeline to obtain measurements of their size and shape. Next, we confirmed the expression of different MN markers at the transcript and protein levels by qPCR and immunocytochemistry of tissue-cleared samples, respectively. Finally, we assessed the capacity of the MN spheroids to display functional activity in the form of action potentials and bursts using a microelectrode array approach. Although most of the cells displayed an MN identity, we also characterized the presence of other cell types, namely interneurons and oligodendrocytes, which share the same neural progenitor pool with MNs. In summary, we successfully developed an MN 3D model, and we optimized a workflow that can be applied to perform its morphological, gene expression, protein, and functional profiling over time.

Developmental trajectories of thalamic progenitors revealed by single-cell transcriptome profiling and Shh perturbation.

The thalamus is the principal information hub of the vertebrate brain, with essential roles in sensory and motor information processing, attention, and memory. The complex array of thalamic nuclei develops from a restricted pool of neural progenitors. We apply longitudinal single-cell RNA sequencing and regional abrogation of Sonic hedgehog (Shh) to map the developmental trajectories of thalamic progenitors, intermediate progenitors, and post-mitotic neurons as they coalesce into distinct thalamic nuclei. These data reveal that the complex architecture of the thalamus is established early during embryonic brain development through the coordinated action of four cell differentiation lineages derived from Shh-dependent and -independent progenitors. We systematically characterize the gene expression programs that define these thalamic lineages across time and demonstrate how their disruption upon Shh depletion causes pronounced locomotor impairment resembling infantile Parkinson's disease. These results reveal key principles of thalamic development and provide mechanistic insights into neurodevelopmental disorders resulting from thalamic dysfunction.

Biphasic cell cycle defect causes impaired neurogenesis in down syndrome.

Impaired neurogenesis in Down syndrome (DS) is characterized by reduced neurons, increased glial cells, and delayed cortical lamination. However, the underlying cause for impaired neurogenesis in DS is not clear. Using both human and mouse iPSCs, we demonstrate that DS impaired neurogenesis is due to biphasic cell cycle dysregulation during the generation of neural progenitors from iPSCs named the “neurogenic stage” of neurogenesis. Upon neural induction, DS cells showed reduced proliferation during the early phase followed by increased proliferation in the late phase of the neurogenic stage compared to control cells. While reduced proliferation in the early phase causes reduced neural progenitor pool, increased proliferation in the late phase leads to delayed post mitotic neuron generation in DS. RNAseq analysis of late-phase DS progenitor cells revealed upregulation of S phase-promoting regulators, Notch, Wnt, Interferon pathways, and REST, and downregulation of several genes of the BAF chromatin remodeling complex. NFIB and POU3F4, neurogenic genes activated by the interaction of PAX6 and the BAF complex, were downregulated in DS cells. ChIPseq analysis of late-phase neural progenitors revealed aberrant PAX6 binding with reduced promoter occupancy in DS cells. Together, these data indicate that impaired neurogenesis in DS is due to biphasic cell cycle dysregulation during the neurogenic stage of neurogenesis.

Autism-associated CHD8 keeps proliferation of human neural progenitors in check by lengthening the G1 phase of the cell cycle.

ABSTRACTDe novo mutations (DNMs) in chromodomain helicase DNA binding protein 8 (CHD8) are associated with a specific subtype of autism characterized by enlarged heads and distinct cranial features. The vast majority of these DNMs are heterozygous loss-of-function mutations with high penetrance for autism. CHD8 is a chromatin remodeler that preferentially regulates expression of genes implicated in early development of the cerebral cortex. How CHD8 haploinsufficiency alters the normal developmental trajectory of the brain is poorly understood and debated. Using long-term single-cell imaging, we show that disruption of a single copy of CHD8 in human neural precursor cells (NPCs) markedly shortens the G1 phase of the cell cycle. Consistent with faster progression of CHD8+/− NPCs through G1 and the G1/S checkpoint, we observed increased expression of E cyclins and elevated phosphorylation of Erk in these mutant cells – two central signaling pathways involved in S phase entry. Thus, CHD8 keeps proliferation of NPCs in check by lengthening G1, and mono-allelic disruption of this gene alters cell-cycle timing in a way that favors self-renewing over neurogenic cell divisions. Our findings further predict enlargement of the neural progenitor pool in CHD8+/− developing brains, providing a mechanistic basis for macrocephaly in this autism subtype.

Fat3 regulates neural progenitor cells by promoting Yap activity during spinal cord development

Early embryonic development of the spinal cord requires tight coordination between proliferation of neural progenitors and their differentiation into distinct neuronal cell types to establish intricate neuronal circuits. The Hippo pathway is one of the well-known regulators to control cell proliferation and govern neural progenitor cell number, in which the downstream effector Yes-associated protein (Yap) promotes cell cycle progression. Here we show that an atypical cadherin Fat3, expressed highly in the neural tube, plays a critical role in maintaining proper number of proliferating progenitors. Knockdown of Fat3 in chick neural tube down-regulates expression of the proliferation markers but rather induces the expression of neural markers in the ventricular zone. We further show that deletion of Fat3 gene in mouse neural tube depletes neural progenitors, accompanied by neuronal gene expression in the ventral ventricular zone of the spinal cord. Finally, we found that Fat3 regulates the phosphorylation level of Lats1/2, the upstream kinase of Yap, resulting in dephosphorylation and stabilization of Yap, suggesting Yap as a key downstream effector of Fat3. Our study uncovers another layer of regulatory mechanisms in controlling the activity of Hippo signaling pathway to regulate the size of neural progenitor pools in the developing spinal cord.

An epigenetic circuit controls neurogenic programs during neocortex development.

The production and expansion of intermediate progenitors (IPs) are essential for neocortical neurogenesis during development and over evolution. Here, we have characterized an epigenetic circuit that precisely controls neurogenic programs, particularly properties of IPs, during neocortical development. The circuit comprises a long non-coding RNA (LncBAR) and the BAF (SWI/SNF) chromatin-remodeling complex, which transcriptionally maintains the expression of Zbtb20. LncBAR knockout neocortex contains more deep-layer but fewer upper-layer projection neurons. Intriguingly, loss of LncBAR promotes IP production, but paradoxically prolongs the duration of the cell cycle of IPs during mid-later neocortical neurogenesis. Moreover, in LncBAR knockout mice, depletion of the neural progenitor pool at embryonic stage results in fewer adult neural progenitor cells in the subventricular zone of lateral ventricles, leading to a failure in adult neurogenesis to replenish the olfactory bulb. LncBAR binds to BRG1, the core enzymatic component of the BAF chromatin-remodeling complex. LncBAR depletion enhances association of BRG1 with the genomic locus of, and suppresses the expression of, Zbtb20, a transcription factor gene known to regulate both embryonic and adult neurogenesis. ZBTB20 overexpression in LncBAR-knockout neural precursors reverses compromised cell cycle progressions of IPs.

Diversity in striatal synaptic circuits arises from distinct embryonic progenitor pools in the ventral telencephalon.

SummarySynaptic circuits in the brain are precisely organized, but the processes that govern this precision are poorly understood. Here, we explore how distinct embryonic neural progenitor pools in the lateral ganglionic eminence contribute to neuronal diversity and synaptic circuit connectivity in the mouse striatum. In utero labeling of Tα1-expressing apical intermediate progenitors (aIP), as well as other progenitors (OP), reveals that both progenitors generate direct and indirect pathway spiny projection neurons (SPNs) with similar electrophysiological and anatomical properties and are intermingled in medial striatum. Subsequent optogenetic circuit-mapping experiments demonstrate that progenitor origin significantly impacts long-range excitatory input strength, with medial prefrontal cortex preferentially driving aIP-derived SPNs and visual cortex preferentially driving OP-derived SPNs. In contrast, the strength of local inhibitory inputs among SPNs is controlled by birthdate rather than progenitor origin. Combined, these results demonstrate distinct roles for embryonic progenitor origin in shaping neuronal and circuit properties of the postnatal striatum.

Ablation of Vti1a/1b Triggers Neural Progenitor Pool Depletion and Cortical Layer 5 Malformation in Late-embryonic Mouse Cortex

Cortical morphogenesis entails several neurobiological events, including proliferation and differentiation of progenitors, migration of neuroblasts, and neuronal maturation leading to functional neural circuitry. These neurodevelopmental processes are delicately regulated by many factors. Endosomal SNAREs have emerged as formidable modulators of neuronal growth, aside their well-known function in membrane/vesicular trafficking. However, our understanding of their influence on cortex formation is limited. Here, we report that the SNAREs Vti1a and Vti1b (Vti1a/1b) are critical for proper cortical development. Following null mutation of Vti1a/1b in mouse, the late-embryonic mutant cortex appeared dysgenic, and the cortical progenitors therein were depleted beyond normal. Notably, cortical layer 5 (L5) is distinctively disorganized in the absence of Vti1a/1b. The latter defect, coupled with an overt apoptosis of Ctip2-expressing L5 neurons, likely contributed to the substantial loss of corticospinal and callosal projections in the Vti1a/1b-deficient mouse brain. These findings suggest that Vti1a/1b serve key neurodevelopmental functions during cortical histogenesis, which when mechanistically elucidated, can lend clarity to how endosomal SNAREs regulate brain development, or how their dysfunction may have implications for neurological disorders.

Stage-dependent effects of intermittent hypoxia influence the outcome of hippocampal adult neurogenesis

Over one billion adults worldwide are estimated to suffer from sleep apnea, a condition with wide-reaching effects on brain health. Sleep apnea causes cognitive decline and is a risk factor for neurodegenerative conditions such as Alzheimer’s disease. Rodents exposed to intermittent hypoxia (IH), a hallmark of sleep apnea, exhibit spatial memory deficits associated with impaired hippocampal neurophysiology and dysregulated adult neurogenesis. We demonstrate that IH creates a pro-oxidant condition that reduces the Tbr2+ neural progenitor pool early in the process, while also suppressing terminal differentiation of adult born neurons during late adult neurogenesis. We further show that IH-dependent cell-autonomous hypoxia inducible factor 1-alpha (HIF1a) signaling is activated in early neuroprogenitors and enhances the generation of adult born neurons upon termination of IH. Our findings indicate that oscillations in oxygen homeostasis, such as those found in sleep apnea, have complex stage-dependent influence over hippocampal adult neurogenesis.

Progenitor cell diversity in the developing mouse neocortex

In the mammalian neocortex, projection neuron types are sequentially generated by the same pool of neural progenitors. How neuron type specification is related to developmental timing remains unclear. To determine whether temporal gene expression in neural progenitors correlates with neuron type specification, we performed single-cell RNA sequencing (scRNA-Seq) analysis of the developing mouse neocortex. We uncovered neuroepithelial cell enriched genes such as Hmga2 and Ccnd1 when compared to radial glial cells (RGCs). RGCs display dynamic gene expression over time; for instance, early RGCs express higher levels of Hes5, and late RGCs show higher expression of Pou3f2 Interestingly, intermediate progenitor cell marker gene Eomes coexpresses temporally with known neuronal identity genes at different developmental stages, though mostly in postmitotic cells. Our results delineate neural progenitor cell diversity in the developing mouse neocortex and support that neuronal identity genes are transcriptionally evident in Eomes-positive cells.

Distinct Embryonic Progenitor Pools in the Ventral Telencephalon Generate Fine-Scale Striatal Synaptic Circuits

Synaptic circuits in the brain are precisely organized, but the processes that govern this precision are poorly understood. Here we explored how distinct embryonic neural progenitor pools in the lateral ganglionic eminence (LGE) contribute to the synaptic circuit connectivity and neuronal diversity in the postnatal mouse striatum. In utero labeling of apical intermediate progenitors (aIP), as well as other progenitors (OP), revealed that both generate spiny projection neurons (SPNs), consisting of D1-expressing direct pathway (dSPNs) as well as D2-expressing indirect pathway (iSPNs) found intermingled in the medial aspects of striatum, with similar electrophysiological and anatomical properties. Subsequent optogenetic circuit-mapping of local synaptic connections between these SPNs showed that neurogenic stage rather than progenitor origin controls the strength of lateral GABAergic connections. However, embryonic progenitor origin conveyed strong biases in their long-range cortical inputs, in that aIP-derived SPNs were preferentially driven by inputs from the medial prefrontal cortex whereas OP-derived SPNs were strongly driven by inputs from the visual cortex. Combined, these results demonstrate critical roles for embryonic origin in shaping the inhibitory and excitatory synaptic circuits in the striatum.

Changes in Cellular Composition of Dentate Gyrus in Response to Intermittent Hypoxia

Adult neurogenesis allows for the continual generation of neurons throughout adulthood. It is implicated in the maintenance of normal hippocampal function and is tightly regulated by oxygen homeostasis. Sleep apnea causes periodic cessations in breathing and leads to intermittent hypoxia (IH), which can impair hippocampal based learning and memory. This ongoing study examines how IH influences neural precursors and remodels the neurogenic niche in the hippocampus. We hypothesize that IH destabilizes the pool of intermediate neural progenitors (INPs) and recruits microglia into the area. Our findings indicate that the number of INPs is reduced by IH. Moreover, while IH does not change the number of microglia in the molecular and granular layers of the dentate gyrus, microglia appear to be uniquely recruited into the neurogenic niche (i.e., the subgranular zone) following IH. These findings suggest that IH‐dependent changes in hippocampal adult neurogenesis may stimulate microglial activity which in turn further impact normal hippocampal function. Such effects may play a significant role in influencing hippocampal physiology during untreated sleep apnea.Support or Funding InformationThis work was supported by NIH PO 1 HL 144454, NIH R01 NS10742101 awarded to AJG, and a grant from The BSD Office of Diversity &amp; Inclusion at The University of Chicago awarded to AJG.

Delta-Notch Signaling: The Long and The Short of a Neuron's Influence on Progenitor Fates.

Maintenance of the neural progenitor pool during embryonic development is essential to promote growth of the central nervous system (CNS). The CNS is initially formed by tightly compacted proliferative neuroepithelial cells that later acquire radial glial characteristics and continue to divide at the ventricular (apical) and pial (basal) surface of the neuroepithelium to generate neurons. While neural progenitors such as neuroepithelial cells and apical radial glia form strong connections with their neighbours at the apical and basal surfaces of the neuroepithelium, neurons usually form the mantle layer at the basal surface. This review will discuss the existing evidence that supports a role for neurons, from early stages of differentiation, in promoting progenitor cell fates in the vertebrates CNS, maintaining tissue homeostasis and regulating spatiotemporal patterning of neuronal differentiation through Delta-Notch signalling.

A Highly Conserved Circular RNA Is Required to Keep Neural Cells in a Progenitor State in the Mammalian Brain

circSLC45A4 is the main RNA splice isoform produced from its genetic locus and one of the highest expressed circRNAs in the developing human frontal cortex. Knockdown of this highly conserved circRNA in a human neuroblastoma cell line is sufficient toinduce spontaneous neuronal differentiation, measurable by increased expression of neuronal marker genes. Depletion of circSlc45a4 in the developing mouse cortex causes a significant reduction of the basal progenitor pool and increases the expression of neurogenic regulators. Furthermore, knockdown of circSlc45a4a induces a significant depletion of cells in the cortical plate. In addition, deconvolution of the bulk RNA-seq data with the help of single-cell RNA-seq data validates the depletion of basal progenitors and reveals an increase in Cajal-Retzius cells. In summary, we present a detailed study of a highly conserved circular RNA that is necessary to maintain the pool of neural progenitors invitro and invivo.

Contributions of Single-Cell Approaches for Probing Heterogeneity and Dynamics of Neural Progenitors Throughout Life: Concise Review.

Development of the forebrain occurs in a stepwise manner from a pool of neural progenitors (NPs), which differs over space and time to produce distinct progenies. The sequence of events leading to the generation of the exquisite complexity of cell types that compose this tissue has been described in great detail at the population level. Recent advances in histology and transcriptomics have allowed probing spatial and temporal heterogeneity and dynamics of NPs at the single-cell level. Clonal fate mapping studies highlight a deterministic behavior as well as the existence of trajectories in the lineage progression of prenatal and postnatal NPs, whereas single-cell transcriptomic studies shed new light on the transcriptional signatures of these processes. Here, we review this recent work and integrate it to our current understanding of forebrain germinal activity at prenatal and postnatal time points. Stem Cells 2019;37:1381-1388.

Zeb1 is important for proper cleavage plane orientation of dividing progenitors and neuronal migration in the mouse neocortex.

During neocortical development, there are two important events, including expansion of the neural progenitor pool through symmetric divisions, and generation of neurons via asymmetrical divisions that lead to a serial process of neuronal polarization, migration, and layer-type specific phenotype acquisition. The mechanisms underlying these processes remain poorly elucidated. Here, we show that the transcription factor Zeb1 regulates the orientation of the cleavage plane of dividing neural progenitors, neuronal polarity, and migration. Upon Zeb1 removal, the cleavage plane of mitotic neural progenitors fails to orientate vertically, resulting in random orientation and premature neuronal differentiation. Consequently, these extra number of precociously produced neurons migrate aberrantly to the upper layer. Mechanistically, we show that Zeb1 suppresses Pak3, a p21-activated serine/threonine protein kinase, through formation of a functional repressing complex together with methyltransferase PRMT5 and Pak3. Our results reveal that Zeb1 plays an essential role in neocortical development and may provide insights into the mechanisms responsible for cortical developmental diseases.