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The cingulate gyrus, as a prominent part of the human limbic lobe, is involved in the integration and regulation of complex emotional, executive, motivational, and cognitive functions, attributed to several functional regions along the anteroposterior axis. In contrast to increasing knowledge of cingulate function in the adult brain, our knowledge of cingulate development is based primarily on classical neuroembryological studies. We aimed to reveal the laminar and cellular development of the various cingulate regions during the critical period from 7.5 to 15 postconceptional weeks (PCW) before the formation of Brodmann type arealization, employing diverse molecular markers on serial histological sections of postmortem human fetal brains. The study was performed by analysis of: (1) deep projection neuron (DPN) markers laminar dynamics, (2) all transient laminar compartments, and (3) characteristic subplate (SP) formation-expansion phase. We found that DPN markers labeling an incipient cortical plate (CP) were the first sign of regional differentiation of the dorsal isocortical and ventral mesocortical belt. Remarkably, increased width of the fibrillar marginal zone (MZ) towards the limbus, in parallel with the narrowing of CP containing DPN, as well as the diminishment of subventricular zone (SVZ) were reliable landmarks of early mesocortical differentiation. Finally, the SP formation pattern was shown to be a crucial event in the isocortical cingulate portion, given that the mesocortical belt is characterized by an incomplete CP delamination and absence of SP expansion. In conclusion, laminar DPN markers dynamics, together with the SVZ size and mode of SP formation indicate regional belt-like cingulate cortex differentiation before the corpus callosum expansion and several months before Brodmann type arealization.

Little is known about the development of the human entorhinal cortex (EC), a major hub in a widespread network for learning and memory, spatial navigation, high-order processing of object information, multimodal integration, attention and awareness, emotion, motivation, and perception of time. We analyzed a series of 20 fetal and two adult human brains using Nissl stain, acetylcholinesterase (AChE) histochemistry, and immunocytochemistry for myelin basic protein (MBP), neuronal nuclei antigen (NeuN), a pan-axonal neurofilament marker, and synaptophysin, as well as postmortem 3T MRI. In comparison with other parts of the cerebral cortex, the cytoarchitectural differentiation of the EC begins remarkably early, in the 10th week of gestation (w.g.). The differentiation occurs in a superficial magnocellular layer in the deep part of the marginal zone, accompanied by cortical plate (CP) condensation and multilayering of the deep part of CP. These processes last until the 13-14th w.g. At 14 w.g., the superficial lamina dissecans (LD) is visible, which divides the CP into the lamina principalis externa (LPE) and interna (LPI). Simultaneously, the rostral LPE separates into vertical cell-dense islands, whereas in the LPI, the deep LD emerges as a clear acellular layer. In the 16th w.g., the LPE remodels into vertical cell-dense and cell-sparse zones with a caudorostral gradient. At 20 w.g., NeuN immunoreactivity is most pronounced in the islands of layer II cells, whereas migration and differentiation inside-out gradients are seen simultaneously in both the upper (LPE) and the lower (LPI) pyramidal layers. At this stage, the EC adopts for the first time an adult-like cytoarchitectural organization, the superficial LD becomes discernible by 3T MRI, MBP- expressing oligodendrocytes first appear in the fimbria and the perforant path (PP) penetrates the subiculum to reach its molecular layer and travels along through the Cornu Ammonis fields to reach the suprapyramidal blade of the dentate gyrus, whereas the entorhinal-dentate branch perforates the hippocampal sulcus about 2-3 weeks later. The first AChE reactivity appears as longitudinal stripes at 23 w.g. in layers I and II of the rostrolateral EC and then also as AChE-positive in-growing fibers in islands of superficial layer III and layer II neurons. At 40 w.g., myelination of the PP starts as patchy MBP-immunoreactive oligodendrocytes and their processes. Our results refute the possibility of an inside-out pattern of the EC development and support the key role of layer II prospective stellate cells in the EC lamination. As the early cytoarchitectural differentiation of the EC is paralleled by the neurochemical development, these developmental milestones in EC structure and connectivity have implications for understanding its normal function, including its puzzling modular organization and potential contribution to consciousness content (awareness), as well as for its insufficiently explored deficits in developmental, psychiatric, and degenerative brain disorders.

The neostriatum plays a central role in cortico-subcortical circuitry underlying goal-directed behavior. The adult mammalian neostriatum shows chemical and cytoarchitectonic compartmentalization in line with the connectivity. However, it is poorly understood how and when fetal compartmentalization (AChE-rich islands, nonreactive matrix) switches to adult (AChE-poor striosomes, reactive matrix) and how this relates to the ingrowth of corticostriatal afferents. Here, we analyze neostriatal compartments on postmortem human brains from 9 postconceptional week (PCW) to 18 postnatal months (PM), using Nissl staining, histochemical techniques (AChE, PAS-Alcian), immunohistochemistry, stereology, and comparing data with volume-growth of in vivo and in vitro MRI. We find that compartmentalization (C) follows a two-compartment (2-C) pattern around 10PCW and is transformed into a midgestational labyrinth-like 3-C pattern (patches, AChE-nonreactive perimeters, matrix), peaking between 22 and 28PCW during accelerated volume-growth. Finally, compartmentalization resolves perinatally, by the decrease in transient “AChE-clumping,” disappearance of AChE-nonreactive, ECM-rich perimeters, and an increase in matrix reactivity. The initial “mature” pattern appears around 9 PM. Therefore, transient, a 3-C pattern and accelerated neostriatal growth coincide with the expected timing of the nonhomogeneous distribution of corticostriatal afferents. The decrease in growth-related AChE activity and transfiguration of corticostriatal terminals are putative mechanisms underlying fetal compartments reorganization. Our findings serve as normative for studying neurodevelopmental disorders.

Human neurodevelopment is characterized by the appearance, development, and disappearance or transformation of various transient structures that underlie the establishment of connectivity within and between future cortical and subcortical areas. Examples of transient structures in the forebrain (among many others) include the subpial granular layer and the subplate zone. We have previously characterized the precise spatiotemporal dynamics of microglia in the human telencephalon. Here, we describe the diversity of microglial morphologies in the subpial granular layer and the subplate zone. Where possible, we couple the predominant morphological phenotype with functional characterizations to infer tentative roles for microglia in a changing neurodevelopmental landscape. We interpret these findings within the context of relevant morphogenetic and neurogenetic events in humans. Due to the unique genetic, molecular, and anatomical features of the human brain and because many human neurological and psychiatric diseases have their origins during development, these structures deserve special attention.

Cytoarchitectonical parcellation of the visual cortex into the striate and extrastriate cortex requires complex histogenetic events within a precise spatio-temporal frame to attain the specification of areal domains and associated thalamocortical connections during the fetal brain development. We analyzed a deep subplate cellular monolayer (subplate “corridor” cells) present during a restricted period of 13–15 postconceptional weeks, showing the 3D caudo-ventro-medial position in the human fetal occipital lobe, corresponding to the segregation point of pulvinocortical and geniculocortical fibers at the prospective area 17/18 border. Immunofluorescence stainings revealed subplate “corridor” cells as the specific class of the deepest subplate neurons (NeuN+, Tbr1+, Cplx3+) expressing axon guidance molecules (Sema-3A+, EphA6+), presumably for the attraction of pulvinocortical axons and the repulsion of geniculocortical axons growing at that time (SNAP25+, Syn+, FN+). Furthermore, quantitative analysis of the subplate “corridor” region of interest, considering cell number, immunofluorescence signal intensity per cell and per region, revealed significant differences to other regions across the tangential circumference of the developing cerebral wall. Thus, our study sheds new light on the deepest subplate sublayer, strategically aligned along the growing axon systems in the prospective visual system, suggesting the establishment of the area 17/18 border by differential thalamocortical input during the fetal brain development.

During the early postnatal period, cerebral white matter undergoes rapid maturation through a complex series of interrelated cellular and histogenetic processes. Accurately quantifying these processes is important for improving understanding of early brain development, developmental abnormalities related to prematurity, and neurodevelopmental diseases. Past efforts have used magnetic resonance imaging (MRI) to track these developmental processes in vivo. However, most previous studies have relied on single imaging modality data and have often been limited by small samples and analytics that do not evaluate complex multivariate imaging patterns. Here, we applied an advanced unsupervised multivariate pattern analysis technique, non-negative matrix factorization (NMF), to T2w/T1w signal ratio maps from a large cohort of newborns (Developing Human Connectome Project [dHCP], n=342), revealing patterns of synchronous white matter maturation. These patterns showed divergent age-related maturational trajectories and differential susceptibility to premature birth, which were replicated in an independent large sample of newborns (Early Life Adversity, Biological Embedding, and Risk for Developmental Precursors of Mental Disorders [eLABE], n=239). Furthermore, we showed that T2w/T1w signal variations in white matter maturational patterns are explained by differential contributions of white matter microstructure indices (i.e., free water content and neurite density index) derived from neurite orientation dispersion and density imaging (NODDI) modeling of diffusion-weighted MRI. Finally, we demonstrated how white matter maturation patterns relate to distinct histological features by comparing our findings with postmortem late fetal/early postnatal brain tissue staining. Together, these results delineate a novel MRI representation of white matter microstructural and histological reorganization during the early postnatal development.

The granular dorsolateral prefrontal cortex (dlPFC) is an evolutionary specialization of primates that is centrally involved in cognition. We assessed more than 600,000 single-nucleus transcriptomes from adult human, chimpanzee, macaque, and marmoset dlPFC. Although most cell subtypes defined transcriptomically are conserved, we detected several that exist only in a subset of species as well as substantial species-specific molecular differences across homologous neuronal, glial, and non-neural subtypes. The latter are exemplified by human-specific switching between expression of the neuropeptide somatostatin and tyrosine hydroxylase, the rate-limiting enzyme in dopamine production in certain interneurons. The above molecular differences are also illustrated by expression of the neuropsychiatric risk gene FOXP2 , which is human-specific in microglia and primate-specific in layer 4 granular neurons. We generated a comprehensive survey of the dlPFC cellular repertoire and its shared and divergent features in anthropoid primates.

The neural crest hypothesis states that the phenotypic features of the domestication syndrome are due to a reduced number or disruption of neural crest cells (NCCs) migration, as these cells differentiate at their final destinations and proliferate into different tissues whose activity is reduced by domestication. Comparing the phenotypic characteristics of modern and prehistoric man, it is clear that during their recent evolutionary past, humans also went through a process of self-domestication with a simultaneous prolongation of the period of socialization. This has led to the development of social abilities and skills, especially language, as well as neoteny. Disorders of neural crest cell development and migration lead to many different conditions such as Waardenburg syndrome, Hirschsprung disease, fetal alcohol syndrome, DiGeorge and Treacher-Collins syndrome, for which the mechanisms are already relatively well-known. However, for others, such as Williams-Beuren syndrome and schizophrenia that have the characteristics of hyperdomestication, and autism spectrum disorders, and 7dupASD syndrome that have the characteristics of hypodomestication, much less is known. Thus, deciphering the biological determinants of disordered self-domestication has great potential for elucidating the normal and disturbed ontogenesis of humans, as well as for the understanding of evolution of mammals in general.

During the second half of gestation, the human cerebrum undergoes pivotal histogenetic events that underlie functional connectivity. These include the growth, guidance, selection of axonal pathways, and their first engagement in neuronal networks. Here, we characterize the spatiotemporal patterns of cerebral connectivity in extremely preterm (EPT), very preterm (VPT), preterm and term babies, focusing on magnetic resonance imaging (MRI) and histological data. In the EPT and VPT babies, thalamocortical axons enter into the cortical plate creating the electrical synapses. Additionally, the subplate zone gradually resolves in the preterm and term brain in conjunction with the growth of associative pathways leading to the activation of large-scale neural networks. We demonstrate that specific classes of axonal pathways within cerebral compartments are selectively vulnerable to temporally nested pathogenic factors. In particular, the radial distribution of axonal lesions, that is, radial vulnerability, is a robust predictor of clinical outcome. Furthermore, the subplate tangential nexus that we can visualize using MRI could be an additional marker as pivotal in the development of cortical connectivity. We suggest to direct future research toward the identification of sensitive markers of earlier lesions, the elucidation of genetic mechanisms underlying pathogenesis, and better long-term follow-up using structural and functional MRI.

Cut-Like Homeobox 2 (Cux2) is a transcription factor involved in dendrite and spine development, and synapse formation of projection neurons placed in mouse upper neocortical layers. Therefore, Cux2 is often used as an upper layer marker in the mouse brain. However, expression of its orthologue CUX2 remains unexplored in the human fetal neocortex. Here, we show that CUX2 protein is expressed in transient compartments of developing neocortical anlage during the main fetal phases of neocortical laminar development in human brain. During the early fetal phase when neurons of the upper cortical layers are still radially migrating to reach their final place in the cortical anlage, CUX2 was expressed in the marginal zone (MZ), deep cortical plate, and pre-subplate. During midgestation, CUX2 was still expressed in the migrating upper cortical neurons as well as in the subplate (SP) and MZ neurons. At the term age, CUX2 was expressed in the gyral white matter along with its expected expression in the upper layer neurons. In sum, CUX2 was expressed in migratory neurons of prospective superficial layers and in the diverse subpopulation of transient postmigratory SP and MZ neurons. Therefore, our findings indicate that CUX2 is a novel marker of distinct transient, but critical histogenetic events during corticogenesis. Given the Cux2 functions reported in animal models, our data further suggest that the expression of CUX2 in postmigratory SP and MZ neurons is associated with their unique dendritic and synaptogenesis characteristics.

A population of more than six million people worldwide at high risk of Alzheimer’s disease (AD) are those with Down Syndrome (DS, caused by trisomy 21 (T21)), 70% of whom develop dementia during lifetime, caused by an extra copy of β-amyloid-(Aβ)-precursor-protein gene. We report AD-like pathology in cerebral organoids grown in vitro from non-invasively sampled strands of hair from 71% of DS donors. The pathology consisted of extracellular diffuse and fibrillar Aβ deposits, hyperphosphorylated/pathologically conformed Tau, and premature neuronal loss. Presence/absence of AD-like pathology was donor-specific (reproducible between individual organoids/iPSC lines/experiments). Pathology could be triggered in pathology-negative T21 organoids by CRISPR/Cas9-mediated elimination of the third copy of chromosome 21 gene BACE2, but prevented by combined chemical β and γ-secretase inhibition. We found that T21 organoids secrete increased proportions of Aβ-preventing (Aβ1–19) and Aβ-degradation products (Aβ1–20 and Aβ1–34). We show these profiles mirror in cerebrospinal fluid of people with DS. We demonstrate that this protective mechanism is mediated by BACE2-trisomy and cross-inhibited by clinically trialled BACE1 inhibitors. Combined, our data prove the physiological role of BACE2 as a dose-sensitive AD-suppressor gene, potentially explaining the dementia delay in ~30% of people with DS. We also show that DS cerebral organoids could be explored as pre-morbid AD-risk population detector and a system for hypothesis-free drug screens as well as identification of natural suppressor genes for neurodegenerative diseases.

Sagittal strata represent bilateral, parasagittally oriented, white matter fiber system running from the frontal towards the occipital pole. They appear during the early fetal life, form trajectories of commissural, projection, and associative axonal pathways, and participate in the transformation of transient fetal zones/white matter segments into the fetal white matter during the late fetal period. Early sagittal strata (10 PCW) appear in the intermediate zone containing thalamic projection and fibers from basal forebrain. During the mid-gestation, they show complex histogenetic “interaction” with adjacent fetal (mainly proliferative) zones, forming multilaminar architecture of fibrillar and cellular layers. Approaching the late fetal period, associative pathways develop at the border of sagittal strata and deep portion of the subplate zone, emphasizing their potential clinical meaning, especially considering their prominent display on magnetic resonance imaging.

A population of >6 million people worldwide at high risk of Alzheimer’s disease (AD) are those with Down Syndrome (DS, caused by trisomy 21 (T21)), 70% of whom develop dementia during lifetime, caused by an extra copy of β-amyloid-(Aβ)-precursor-protein gene. We report AD-like pathology in cerebral organoids grownin vitrofrom non-invasively sampled strands of hair from 71% of DS donors. The pathology consisted of extracellular diffuse and fibrillar Aβ deposits, hyperphosphorylated/pathologically conformed Tau, and premature neuronal loss.Presence/absence of AD-like pathology was donor-specific (reproducible between individual organoids/iPSC lines/experiments). Pathology could be triggered in pathology-negative T21 organoids by CRISPR/Cas9-mediated elimination of the third copy of chromosome-21-geneBACE2, but prevented by combined chemical β and γ-secretase inhibition. We found that T21-organoids secrete increased proportions of Aβ-preventing (Aβ1-19) and Aβ-degradation products (Aβ1-20 and Aβ1-34). We show these profiles mirror in cerebrospinal fluid of people with DS. We demonstrate that this protective mechanism is mediated by BACE2-trisomy and cross-inhibited by clinically trialled BACE1-inhibitors. Combined, our data prove the physiological role ofBACE2as a dose-sensitive AD-suppressor gene, potentially explaining the dementia delay in ∼30% of people with DS. We also show that DS cerebral organoids could be explored as pre-morbid AD-risk population detector and a system for hypothesis-free drug screens as well as identification of natural suppressor genes for neurodegenerative diseases.

Amygdala represents a heterogeneous nuclear complex consisting of morphologically different nuclei involved in various functions regarding emotional modulation. Previously, we have demonstrated a human specific transient modular organization of this structure during the mid- fetal period (Nikolic & Kostović, Anat Embryol 1986). Part of a phylogenetically older complex, central amygdaloid nucleus represents the main efferent structure projecting to many brain regions, mainly via stria terminalis and ventral amygdalofugal pathway. Here we studied possible indicators of the underlining molecular mechanisms in regional differences in maturational processes of this nuclear complex. Reviewing publicly available gene expression database of human brain (Kang et al., Nature 2011), several genes were selected which are specifically and highly expressed in human amygdala prenatally. Among them is a transcription factor Dlx6, required for molecular differentiation of striatal neurons in developing mouse brain (Wang et al, J. Comp. Neurol. 2011). To investigate DLX6 expression, we employed immunohistochemistry on fixed- paraffin-embedded sections of postmortem human brains, ranging between 15th and 28th post conception weeks (PCW). The procedure for the human autopsy material was approved and controlled by the Internal Review Board of the Ethical Committee at the School of Medicine, University of Zagreb. Expression of DLX6 was visible in the amygdaloid primordiums in the prospective central nucleus, revealing intense nuclear staining. Confocal microscopy analysis using double-staining against NeuN and GFAP revealed that DLX6+ cells colocalized with NeuN+ but not with GFAP+ cells, indicating advanced differentiation of central nucleus neurons. Our results indicate that DLX6 could be a part of the regulatory molecular program of amygdaloid regionalization, presumably playing an important role in early differentiation of the central amygdaloid nucleus. Since this nucleus represents the main efferent structure, its advanced maturation supports evidence for very early establishment of amygdaloid visceromotor circuitry.

Secreted frizzled-related protein 3 (SFRP3) is a member of the family of soluble proteins, which modulate the Wnt signaling cascade. Novel research has identified aberrant expression of SFRPs in different types of cancer. In the present study the expression intensities and localizations of the SFRP3 protein across different histopathological grades of astrocytic brain tumors were investigated by immunohistochemistry, digital scanning and image analysis. The results demonstrated that the differences between expression levels and malignancy grades were statistically significant. Tumors were classified into four malignancy grades according to the World Health Organization guidelines. Moderate (P=0.014) and strong (P=0.028) nuclear expression levels were significantly different in pilocytic (grade I) and diffuse (grade II) astrocytomas demonstrating higher expression values, as compared with anaplastic astrocytoma (grade III) and glioblastoma (grade IV). When the sample was divided into two groups, the moderate and high cytoplasmic expression levels were observed to be significantly higher in glioblastomas than in the group comprising astrocytoma II and III. Furthermore, the results indicated that high grade tumors were associated with lower values of moderate (P=0.002) and strong (P=0.018) nuclear expression in comparison to low grade tumors. Analysis of cytoplasmic staining demonstrated that strong cytoplasmic expression was significantly higher in the astrocytoma III and IV group than in the astrocytoma I and II group (P=0.048). Furthermore, lower grade astrocytomas exhibited reduced membranous SFRP3 staining when compared with higher grade astrocytomas and this difference was statistically significant (P=0.036). The present results demonstrated that SFRP3 protein expression levels were decreased in the nucleus in higher grade astrocytoma (indicating the expected behavior of an antagonist of Wnt signaling), whereas when the SFRP3 was located in the cytoplasm an increased expression level of SFRP3 was identified in the high grade astrocytomas when compared with those of a low grade. This may suggest that SFRP3 acts as an agonist of Wnt signaling and promotes invasive behavior.

Key regulators of the Wnt signalling, DVL1, DVL2 and DVL3, in astrocytomas of different malignancy grades were investigated. Markers for DVL1,DVL2 and DVL3 were used to detect microsatellite instability (MSI) and gross deletions (LOH), while immunohistochemistry and immunoreactivity score were used to determine the signal strengths of the three DVL proteins and transcription factors of the pathway, TCF1 and LEF1. Our findings demonstrated that MSI at all three DVL loci was constantly found across tumour grades with the highest number in grade II (P = 0.008). Collectively, LOHs were more frequent in high‐grade tumours than in low grade ones. LOHs of DVL3 gene were significantly associated with grade IV tumours (P = 0.007). The results on protein expressions indicated that high‐grade tumours expressed less DVL1 protein as compared with low grade ones. A significant negative correlation was established between DVL1 expression and malignancy grades (P

Development of the cerebral wall is characterized by partially overlapping histogenetic events. However, little is known with regards to when, where, and how growing axonal pathways interact with progenitor cell lineages in the proliferative zones of the human fetal cerebrum. We analyzed the developmental continuity and spatial distribution of the axonal sagittal strata (SS) and their relationship with proliferative zones in a series of human brains (8-40 post- conceptional weeks ; PCW) by comparing histological, histochemical, and immunocytochemical data with magnetic resonance imaging (MRI). Between 8.5 and 11 PCW, thalamocortical fibers from the intermediate zone (IZ) were initially dispersed throughout the subventricular zone (SVZ), while sizeable axonal "invasion" occurred between 12.5 and 15 PCW followed by callosal fibers which "delaminated" the ventricular zone-inner SVZ from the outer SVZ (OSVZ). During midgestation, the SS extensively invaded the OSVZ, separating cell bands, and a new multilaminar axonal- cellular compartment (MACC) was formed. Preterm period reveals increased complexity of the MACC in terms of glial architecture and the thinning of proliferative bands. The addition of associative fibers and the formation of the centrum semiovale separated the SS from the subplate. In vivo MRI of the occipital SS indicates a "triplet" structure of alternating hypointense and hyperintense bands. Our results highlighted the developmental continuity of sagittally oriented "corridors" of projection, commissural and associative fibers, and histogenetic interaction with progenitors, neurons, and glia. Histogenetical changes in the MACC, and consequently, delineation of the SS on MRI, may serve as a relevant indicator of white matter microstructural integrity in the developing brain.

We describe an as yet unreported neocentric small supernumerary marker chromosome (sSMC) derived from chromosome 1p21.3p21.2. It was present in 80% of the lymphocytes in a male patient with intellectual disability, severe speech deficit, mild dysmorphic features, and hyperactivity with elements of autism spectrum disorder (ASD). Several important neurodevelopmental genes are affected by the 3.56 Mb copy number gain of 1p21.3p21.2, which may be considered reciprocal in gene content to the recently recognized 1p21.3 microdeletion syndrome. Both 1p21.3 deletions and the presented duplication display overlapping symptoms, fitting the same disorder category. Contribution of coding and non-coding genes to the phenotype is discussed in the light of cellular and intercellular homeostasis disequilibrium. In line with this the presented 1p21.3p21.2 copy number gain correlated to 1p21.3 microdeletion syndrome verifies the hypothesis of a cumulative effect of the number of deregulated genes - homeostasis disequilibrium leading to overlapping phenotypes between microdeletion and microduplication syndromes. Although miR-137 appears to be the major player in the 1p21.3p21.2 region, deregulation of the DPYD (dihydropyrimidine dehydrogenase) gene may potentially affect neighboring genes underlying the overlapping symptoms present in both the copy number loss and copy number gain of 1p21. Namely, the all-in approach revealed that DPYD is a complex gene whose expression is epigenetically regulated by long non-coding RNAs (lncRNAs) within the locus. Furthermore, the long interspersed nuclear element-1 (LINE-1) L1MC1 transposon inserted in DPYD intronic transcript 1 (DPYD-IT1) lncRNA with its parasites, TcMAR-Tigger5b and pair of Alu repeats appears to be the “weakest link” within the DPYD gene liable to break. Identification of the precise mechanism through which DPYD is epigenetically regulated, and underlying reasons why exactly the break (FRA1E) happens, will consequently pave the way toward preventing severe toxicity to the antineoplastic drug 5-fluorouracil (5-FU) and development of the causative therapy for the dihydropyrimidine dehydrogenase deficiency.

Thalamocortical (TH-C) fiber growth begins during the embryonic period and is completed by the third trimester of gestation in humans. Here we determined the timing and trajectories of somatosensory TH-C fibers in the developing human brain. We analyzed the periods of TH-C fiber outgrowth, path-finding, “waiting” in the subplate (SP), target selection, and ingrowth in the cortical plate (CP) using histological sections from post-mortem fetal brain [from 7 to 34 postconceptional weeks (PCW)] that were processed with acetylcholinesterase (AChE) histochemistry and immunohistochemical methods. Images were compared with post mortem diffusion tensor imaging (DTI)-based fiber tractography (code No NO1-HD-4-3368). The results showed TH-C axon outgrowth occurs as early as 7.5 PCW in the ventrolateral part of the thalamic anlage. Between 8 and 9.5 PCW, TH-C axons form massive bundles that traverse the diencephalic-telencephalic boundary. From 9.5 to 11 PCW, thalamocortical axons pass the periventricular area at the pallial-subpallial boundary and enter intermediate zone in radiating fashion. Between 12 and 14 PCW, the TH-C axons, aligned along the fibers from the basal forebrain, continue to grow for a short distance within the deep intermediate zone and enter the deep CP, parallel with SP expansion. Between 14 and 18 PCW, the TH-C interdigitate with callosal fibers, running shortly in the sagittal stratum and spreading through the deep SP (“waiting” phase). From 19 to 22 PCW, TH-C axons accumulate in the superficial SP below the somatosensory cortical area; this occurs 2 weeks earlier than in the frontal and occipital cortices. Between 23 and 24 PCW, AChE-reactive TH-C axons penetrate the CP concomitantly with its initial lamination. Between 25 and 34 PCW, AChE reactivity of the CP exhibits an uneven pattern suggestive of vertical banding, showing a basic 6-layer pattern. In conclusion, human thalamocortical axons show prolonged growth (4 months), and somatosensory fibers precede the ingrowth of fibers destined for frontal and occipital areas. The major features of growing TH-C somatosensory fiber trajectories are fan-like radiation, short runs in the sagittal strata, and interdigitation with the callosal system. These results support our hypothesis that TH-C axons are early factors in SP and CP morphogenesis and synaptogenesis and may regulate cortical somatosensory system maturation.

Despite recent impressive advancement in reveling genetic blueprint of the human neocortex, our knowledge on real operation of neural assemblies during cognitive, social, and emotional frontal lobe functions is rather limited. The advent of microarrays and RNA sequencing techniques in the last decade has broadened our knowledge about gene expression in different regions of the human brain throughout whole life span. It enabled us to analyze large-scale changes in the gene expression pattern between various brain regions and in many disorders of the nervous system. The data from these studies significantly advanced our understanding on how human brain develops and functions. However, understanding of the higher cognitive functions requires not only genetic approach but also analysis of interaction of genes and environment in shaping neuronal circuitry and modulation of neural assemblies.

A number of age-related diseases have a low incidence in females, which is attributed to a protective effect of sex hormones. For instance, the female sex hormone estrogen (E2) has a well established cytoprotective effect against oxidative stress, which strongly contributes to ageing. However, the mechanism by which E2 exerts its protective activity remains elusive. In this study we address the question whether the E2-induced protective effect against hyperoxia is mediated by the Nrf-2/Keap-1 signaling pathway. In particular, we investigate the E2-induced expression and cellular distribution of DPP III monozinc exopeptidase, a member of the Nrf-2/Keap-1 pathway, upon hyperoxia treatment. We find that DPP III accumulates in the nucleus in response to hyperoxia. Further, we show that combined induction of hyperoxia and E2 administration have an additive effect on the nuclear accumulation of DPP III. The level of nuclear accumulation of DPP III is comparable to nuclear accumulation of Nrf-2 in healthy female mice exposed to hyperoxia. In ovariectomized females exposed to hyperoxia, supplementation of E2 induced upregulation of DPP III, Ho-1, Sirt-1 and downregulation of Ppar-γ. While other cytoprotective mechanisms cannot be excluded, these findings demonstrate a prominent role of DPP III, along with Sirt-1, in the E2-mediated protection against hyperoxia., Graphical abstract fx1, Highlights • DPP III accumulates in the nucleus in response to hyperoxia. • Additive effect of hyperoxia and E2 on nuclear accumulation of DPP III is observed. • Protective effect of E2 is associated with increased DPP III, Ho-1 and Sirt-1.

Sažetak

Summary Here we report the generation and analysis of genome-wide exon-level transcriptome data from 16 brain regions comprising the cerebellar cortex, mediodorsal nucleus of the thalamus, striatum, amygdala, hippocampus, and 11 areas of the neocortex. The dataset was generated from 1,340 tissue samples collected from one or both hemispheres of 57 postmortem human brains, spanning from embryonic development to late adulthood and representing males and females of multiple ethnicities. We also performed genotyping of 2.5 million SNPs and assessed copy number variations for all donors. Approximately 86% of protein-coding genes were found to be expressed using stringent criteria, and over 90% of these were differentially regulated at the whole transcript or exon level across regions and/or time. The majority of these spatiotemporal differences occurred before birth, followed by an increase in the similarity among regional transcriptomes during postnatal lifespan. Genes were organized into functionally distinct co-expression networks, and sex differences were present in gene expression and exon usage. Finally, we demonstrate how these results can be used to profile trajectories of genes associated with neurodevelopmental processes, cell types, neurotransmitter systems, autism, and schizophrenia, as well as to discover associations between SNPs and spatiotemporal gene expression. This study provides a comprehensive, publicly available dataset on the spatiotemporal human brain transcriptome and new insights into the transcriptional foundations of human neurodevelopment.

SummaryOur understanding of the evolution, formation, and pathological disruption of human brain circuits is impeded by a lack of comprehensive data on the developing brain transcriptome. A whole-genome, exon-level expression analysis of 13 regions from left and right sides of the mid-fetal human brain revealed that 76% of genes are expressed, and 44% of these are differentially regulated. These data reveal a large number of specific gene expression and alternative splicing patterns, as well as coexpression networks, associated with distinct regions and neurodevelopmental processes. Of particular relevance to cognitive specializations, we have characterized the transcriptional landscapes of prefrontal cortex and perisylvian speech and language areas, which exhibit a population-level global expression symmetry. We show that differentially expressed genes are more frequently associated with human-specific evolution of putative cis-regulatory elements. These data provide a wealth of biological insights into the complex transcriptional and molecular underpinnings of human brain development and evolution.

Background: Recent studies have shown remarkable complexity of neuronal differentiation and cellular composition of the marginal zone in the fetal cortex. New immunocytochemical parameters are needed for analysis of dynamic neurogenetic events at the interface between the afferent fibers of the marginal zone and the superficial cortical plate. Aims: The aim of this study was to analyze transient cytoarchitectonic and immunocytochemical patterns of organization of the marginal zone of the human fetal cortex. Results: Fine cytoarchitectural parameters revealed a very complex six-layered organization of the marginal zone of the human fetal cortex between 18 and 28 weeks of gestation (w.g.). Starting from the pia to the cortical plate, the following layers can be distinguished: (1) cell-poor marginal stripe (Randstreifen); (2) subpial granular layer; (3) marginal zone proper; (4) stratum lucidum (SL); (5) deep granular layer (DGL), and (6) stratum radiatum (SR). The subpial granular layer develops around 13 w.g. and is not visible at 34 w.g. The SL, characterized by immunostaining with SNAP-25, MAP1b and SMI 312 antibodies and acetylcholinesterase histochemistry, appears on Nissl sections at around 20 w.g. and disappears after 28 w.g. The DGL contains NeuN and some MAP2-positive cells, presumably neurons, appears at 15 w.g. and disappears by 34 w.g. The SR shows profound transformation in parallel to the changes in the most superficial part of the cortical plate. Conclusions: The marginal zone displays a very complex organization in the human fetal cortex due to the complexity of cortical inputs from both increased population of Cajal-Retzius neurons and abundant afferents to the apical dendrites of cortical plate neurons. Deep transitional sublayers, namely SL, DGL and SR, serve as a compartment for ingrowth of afferents and migratory routes to the superficial part of the cortical plate. A similar developmental role is played by the subplate zone at the deep border of the cortical plate.

Cortical excitatory glutamatergic projection neurons and inhibitory GABAergic interneurons follow substantially different developmental programs. In rodents, projection neurons originate from progenitors within the dorsal forebrain, whereas interneurons arise from progenitors in the ventral forebrain. In contrast, it has been proposed that in humans, the majority of cortical interneurons arise from progenitors within the dorsal forebrain, suggesting that their origin and migration is complex and evolutionarily divergent. However, whether molecularly defined human cortical interneuron subtypes originate from distinct progenitors, including those in the ventral forebrain, remains unknown. Furthermore, abnormalities in cortical interneurons have been linked to human disorders, yet no distinct cell population selective loss has been reported. Here we show that cortical interneurons expressing nitric oxide synthase 1, neuropeptide Y, and somatostatin, are either absent or substantially reduced in fetal and infant cases of human holoprosencephaly (HPE) with severe ventral forebrain hypoplasia. Notably, another interneuron subtype normally abundant from the early fetal period, marked by calretinin expression, and different subtypes of projection neuron were present in the cortex of control and HPE brains. These findings have important implications for the understanding of neuronal pathogenesis underlying the clinical manifestations associated with HPE and the developmental origins of human cortical interneuron diversity.

Neocortical projection neurons exhibit layer-specific molecular profiles and axonal connections. Here we show that the molecular identities of early-born subplate and deep-layer neurons are not acquired solely during generation or shortly thereafter but undergo progressive postmitotic refinement mediated by SOX5. Fezf2 and Bcl11b , transiently expressed in all subtypes of newly postmigratory early-born neurons, are subsequently downregulated in layer 6 and subplate neurons, thereby establishing their layer 5-enriched postnatal patterns. In Sox5 -null mice, this downregulation is disrupted, and layer 6 and subplate neurons maintain an immature differentiation state, abnormally expressing these genes postnatally. Consistent with this disruption, SOX5 binds and represses a conserved enhancer near Fezf2 . The Sox5 -null neocortex exhibits failed preplate partition and laminar inversion of early-born neurons, loss of layer 5 subcerebral axons, and misrouting of subplate and layer 6 corticothalamic axons to the hypothalamus. Thus, SOX5 postmitotically regulates the migration, postmigratory differentiation, and subcortical projections of subplate and deep-layer neurons.

The immunocytochemical distribution of the neuronal form of nitric oxide synthase (nNOS) was compared with neuropathological changes and with cell death related DNA damage (as revealed by in situ end labeling, ISEL) in the hippocampal formation and entorhinal cortex of 12 age-matched control subjects and 12 Alzheimer's disease (AD) patients. Unlike controls, numerous nNOS-positive reactive astrocytes were found in AD patients around beta-amyloid plaques in CA1 and subiculum and at the places of clear and overt neuron loss, particularly in the entorhinal cortex layer II and CA4. This is the first evidence of nNOS-like immunoreactivity in reactive astrocytes in AD. In contrast to controls, in all but one AD subject, large numbers of ISEL-positive neuronal nuclei and microglial cells were found in the CA1 and CA4 regions and subiculum. Semiquantitative analysis showed that neuronal DNA fragmentation in AD match with the distribution of nNOS-expressing reactive astroglial cells in CA1 (r = 0.74, P < 0.01) and CA4 (r = 0.58, P < 0.05). A portion of the nNOS-positive CA2/CA3 pyramidal neurons was found to be spared even in the most affected hippocampi. A significant inverse correlation between nNOS expression and immunoreactivity to abnormally phosphorylated tau proteins (as revealed by AT8 monoclonal antibody) in perikarya of these CA2/3 neurons (r = -0.85, P < 0.01) suggests that nNOS expression may provide selective resistance to neuronal degeneration in AD. In conclusion, our results imply that an upregulated production of NO by reactive astrocytes may play a key role in the pathogenesis of AD.