Transcription, structure, and organoids translate time across the lifespan of humans and great apes.

How the neural structures supporting human cognition developed and arose in evolution is an enduring question of interest. Yet, we still lack appropriate procedures to align ages across primates, and this lacuna has hindered progress in understanding the evolution of biological programs. We generated a dataset of unprecedented size consisting of 573 time points from abrupt and gradual changes in behavior, anatomy, and transcription across human and 8 nonhuman primate species. We included time points from diverse human populations to capture within-species variation in the generation of cross-species age alignments. We also extracted corresponding ages from organoids. The identification of corresponding ages across the lifespan of 8 primate species, including apes (e.g., orangutans, gorillas) and monkeys (i.e., marmosets, macaques), reveals that some biological pathways are extended in humans compared with some nonhuman primates. Notably, the human lifespan is unusually extended relative to studied nonhuman primates demonstrating that very old age is a phase of life in humans that does not map to other studied primate species. More generally, our work prompts a reevaluation in the choice of a model system to understand aging given very old age in humans is a period of life without a clear counterpart in great apes.

Immunofluorescence protocol for localizing protein targets in brain tissue from diverse model and non-model mammals.

Previous immunostaining protocols are highly specific for model organisms and often not suitable for diverse specimens that are non-perfused and over-fixed (i.e., tissues sitting in fixatives for months/year). Here, we present an immunofluorescence protocol for localizing protein targets in brain tissue from 11 model and non-model mammals. We describe preparation of both fresh and fixed tissues including steps for deparaffinization, fixation, and cryoprotection. We then detail immunofluorescence procedures including antigen retrieval, reducing autofluorescence, nuclear staining, mounting, and image collection.

Synaptic boutons are smaller in chandelier cell cartridges in autism.

Chandelier (Ch) cells are cortical interneurons with axon terminal structures known as cartridges that synapse on the axon initial segment of excitatory pyramidal neurons. Previous studies indicate that the number of Ch cells is decreased in autism, and that GABA receptors are decreased in the Ch cell synaptic target in the prefrontal cortex. To further identify Ch cell alterations, we examined whether the length of cartridges, and the number, density, and size of Ch cell synaptic boutons, differed in the prefrontal cortex of cases with autism versus control cases. We collected samples of postmortem human prefrontal cortex (Brodmann Area (BA) 9, 46, and 47) from 20 cases with autism and 20 age- and sex-matched control cases. Ch cells were labeled using an antibody against parvalbumin, a marker that labeles soma, cartridges, and synaptic boutons. We found no significant difference in the average length of cartridges, or in the total number or density of boutons in control subjects vs. subjects with autism. However, we found a significant decrease in the size of Ch cell boutons in those with autism. The reduced size of Ch cell boutons may result in reduced inhibitory signal transmission and impact the balance of excitation to inhibition in the prefrontal cortex in autism.

Spatial control of astrogenesis progression by cortical arealization genes.

Sizes of neuronal, astroglial and oligodendroglial complements forming the neonatal cerebral cortex largely depend on rates at which pallial stem cells give rise to lineage-committed progenitors and the latter ones progress to mature cell types. Here, we investigated the spatial articulation of pallial stem cells' (SCs) commitment to astrogenesis as well as the progression of committed astroglial progenitors (APs) to differentiated astrocytes, by clonal and kinetic profiling of pallial precursors. We found that caudal-medial (CM) SCs are more prone to astrogenesis than rostro-lateral (RL) ones, while RL-committed APs are more keen to proliferate than CM ones. Next, we assessed the control of these phenomena by 2 key transcription factor genes mastering regionalization of the early cortical primordium, Emx2 and Foxg1, via lentiviral somatic transgenesis, epistasis assays, and ad hoc rescue assays. We demonstrated that preferential CM SCs progression to astrogenesis is promoted by Emx2, mainly via Couptf1, Nfia, and Sox9 upregulation, while Foxg1 antagonizes such progression to some extent, likely via repression of Zbtb20. Finally, we showed that Foxg1 and Emx2 may be implicated-asymmetrically and antithetically-in shaping distinctive proliferative/differentiative behaviors displayed by APs in hippocampus and neocortex.

Evolution of astrocytes: From invertebrates to vertebrates.

The central nervous system (CNS) shows incredible diversity across evolution at the anatomical, cellular, molecular, and functional levels. Over the past decades, neuronal cell number and heterogeneity, together with differences in the number and types of neuro-active substances, axonal conduction, velocity, and modes of synaptic transmission, have been rigorously investigated in comparative neuroscience studies. However, astrocytes, a specific type of glial cell in the CNS, play pivotal roles in regulating these features and thus are crucial for the brain's development and evolution. While special attention has been paid to mammalian astrocytes, we still do not have a clear definition of what an astrocyte is from a broader evolutionary perspective, and there are very few studies on astroglia-like structures across all vertebrates. Here, I elucidate what we know thus far about astrocytes and astrocyte-like cells across vertebrates. This information expands our understanding of how astrocytes evolved to become more complex and extremely specialized cells in mammals and how they are relevant to the structure and function of the vertebrate brain.

Decreased number and increased activation state of astrocytes in gray and white matter of the prefrontal cortex in autism.

The cerebral cortex presents with alterations in the number of specific cell types in autism spectrum disorder (ASD). Astrocytes have many functions in the brain including a role in higher cognitive functions and in inflammatory brain processes. Therefore, an alteration in number, function, and/or activation state of astrocytes, could be present in ASD. We quantified astrocyte number in the gray and white matter of the prefrontal cortex-BA9, BA46, and BA47-in 15 ASD and 15 age- and sex-matched control cases. We labeled astrocytes with antibodies against the protein GFAP and S100ß, markers of astrocytes. We found a significant decrease in the number of astrocytes in the gray and white matter of all prefrontal areas of interest with both markers. We also found an increased state of activation of GFAP+ astrocytes in all areas. A reduced number of astrocytes in the cerebral cortex in ASD could lead to impaired synaptic function and disrupted connectivity. An increased astrocyte activation may indicate a chronic mild inflammatory state of the cerebral cortex in ASD. Overall, we found that astrocytes are disrupted in ASD.

Redefining varicose projection astrocytes in primates.

Varicose projection astrocytes (VP-As) are found in the cerebral cortex and have been described to be specific to humans and chimpanzees. To further examine the phylogenetic distribution of this cell type, we analyzed cortical tissue from several primates ranging from primitive primates to primates evolutionary closer to human such as apes. We specifically analyzed tissue from four strepsirrhine species, one tarsier, six species of platyrrhine monkeys, ten species of cercopithecoid monkeys, two hylobatid ape species, four to six cases each of chimpanzee, bonobo, gorilla, and orangutan, and thirteen human. We found that VP-As were present only in human and other apes (hominoids) and were absent in all other species. We showed that VP-As are localized to layer VI and the superficial white matter of the cortex. The presence of VP-As co-occured with interlaminar astrocytes that also had varicosities in their processes. Due to their location, their long tangential processes, and their irregular presence within species, we propose that VP-As are astrocytes that develop varicosities under specific conditions and that are not a distinct astrocyte type.

Neuronal and glial cell number is altered in a cortical layer-specific manner in autism.

LAY ABSTRACT: The cerebral cortex affected with autism spectrum disorder presents changes in the number of neurons and glia cells, possibly leading to a dysregulation of brain circuits and affecting behavior. However, little is known about cell number alteration in specific layers of the cortex in autism spectrum disorder. We found an increase in the number of neurons and a decrease in the number of astrocytes in specific layers of the prefrontal cortex in postmortem human brains from autism spectrum disorder cases. We hypothesize that this may be due to a failure in neural stem cells to shift differentiation from neurons to glial cells during prenatal brain development. These data provide key anatomical findings that contribute to the bases of autism spectrum disorder pathogenesis.

Chandelier Cartridge Density Is Reduced in the Prefrontal Cortex in Autism.

An alteration in the balance of excitation-inhibition has been proposed as a common characteristic of the cerebral cortex in autism, which may be due to an alteration in the number and/or function of the excitatory and/or inhibitory cells that form the cortical circuitry. We previously found a decreased number of the parvalbumin (PV)+ interneuron known as Chandelier (Ch) cell in the prefrontal cortex in autism. This decrease could result from a decreased number of Ch cells, but also from decreased PV protein expression by Ch cells. To further determine if Ch cell number is altered in autism, we quantified the number of Ch cells following a different approach and different patient cohort than in our previous studies. We quantified the number of Ch cell cartridges-rather than Ch cell somata-that expressed GAT1-rather than PV. Specifically, we quantified GAT1+ cartridges in prefrontal areas BA9, BA46, and BA47 of 11 cases with autism and 11 control cases. We found that the density of GAT1+ cartridges was decreased in autism in all areas and layers. Whether this alteration is cause or effect remains unclear but could result from alterations that take place during cortical prenatal and/or postnatal development.

Cortical Interlaminar Astrocytes Are Generated Prenatally, Mature Postnatally, and Express Unique Markers in Human and Nonhuman Primates.

Interlaminar astrocytes (ILAs) are a subset of cortical astrocytes that reside in layer I, express GFAP, have a soma contacting the pia, and contain long interlaminar processes that extend through several cortical layers. We studied the prenatal and postnatal development of ILAs in three species of primates (rhesus macaque, chimpanzee, and human). We found that ILAs are generated prenatally likely from radial glial (RG) cells, that ILAs proliferate locally during gestation, and that ILAs extend interlaminar processes during postnatal stages of development. We showed that the density and morphological complexity of ILAs increase with age, and that ILAs express multiple markers that are expressed by RG cells (Pax6, Sox2, and Nestin), specific to inner and outer RG cells (Cryab and Hopx), and astrocyte markers (S100ß, Aqp4, and GLAST) in prenatal stages and in adult. Finally, we demonstrated that rudimentary ILAs in mouse also express the RG markers Pax6, Sox2, and Nestin, but do not express S100ß, Cryab, or Hopx, and that the density and morphological complexity of ILAs differ between primate species and mouse. Together these findings contribute new information on astrogenesis of this unique class of cells and suggest a lineal relationship between RG cells and ILAs.

GABAARα2 is Decreased in the Axon Initial Segment of Pyramidal Cells in Specific Areas of the Prefrontal Cortex in Autism.

Some forms of Autism Spectrum Disorder, a neurodevelopmental syndrome characterized by impaired communication and social skills as well as repetitive behaviors, are purportedly associated with dysregulation of the excitation/inhibition balance in the cerebral cortex. Through human postmortem tissue analysis, we previously found a significant decrease in the number of a gamma-aminobutyric acid (GABA)ergic interneuron subtype, the chandelier (Ch) cell, in the prefrontal cortex of subjects with autism. Ch cells exclusively target the axon initial segment (AIS) of excitatory pyramidal (Pyr) neurons, and a single Ch cell forms synapses on hundreds of Pyr cells, indicating a possible role in maintaining electrical balance. Thus, we herein investigated this crucial link between Ch and Pyr cells in the anatomy of autism neuropathology by examining GABA receptor protein expression in the Pyr cell AIS in subjects with autism. We collected tissue from the prefrontal cortex (Brodmann Areas (BA) 9, 46, and 47) of 20 subjects with autism and 20 age- and sex-matched control subjects. Immunohistochemical staining with antibodies against the GABAA receptor subunit α2 (GABAARα2) - the subunit most prevalent in the Pyr cell AIS - revealed a significantly decreased GABAARα2 protein in the Pyr cell AIS in supragranular layers of prefrontal cortical areas BA9 and BA47 in autism. Downregulated GABAARα2 protein in the Pyr cell AIS may result from decreased GABA synthesis in the prefrontal cortex of subjects with autism, and thereby contribute to an excitation/inhibition imbalance. Our findings support the potential for GABA receptor agonists asa therapeutic tool for autism.

The fundamental building blocks of cortical development are established in human exencephaly.

BACKGROUND: The presence and status of progenitor/stem cells in excencephalic brain have not been previously examined. METHODS: Brain sections of excencephalic 17-week fetus were stained for specific stem and mature cell markers. RESULTS: The ventricles were open, the developing cerebral cortex was thin in the radial dimension, and the ventricular surface was undulated. There was a decreased ratio of subventricular/ventricular zone radial glia precursor cells (RGCs; PAX6+ and HOPX+ cells), a decreased number of intermediate progenitor cells (IPCs; TBR2+), a decreased number of neurons (MAP2+), and an increased number of astrocytes (S100b+), compared to the control. MAP2+ neurons, S100b+ astrocytes, and OLIG2+ oligodendrocytes were present within the subventricular zone. CONCLUSIONS: This indicates that the underlying condition did not initially preclude radial glial cells from undergoing asymmetric divisions that produce IPCs but halted the developmental progression. RGC and IPC presence in the developing cerebral cortex demonstrates that the fundamental building blocks of cortical formation had been established and that a normal sequence of developmental steps had been initiated in this case of exencephaly. These data expand our understanding of exencephaly etiology and highlight the status of cortical progenitor cells that may be linked to the disorder.

Foxg1 Antagonizes Neocortical Stem Cell Progression to Astrogenesis.

Neocortical astrogenesis follows neuronogenesis and precedes oligogenesis. Among key factors dictating its temporal articulation, there are progression rates of pallial stem cells (SCs) towards astroglial lineages as well as activation rates of astrocyte differentiation programs in response to extrinsic gliogenic cues. In this study, we showed that high Foxg1 SC expression antagonizes astrocyte generation, while stimulating SC self-renewal and committing SCs to neuronogenesis. We found that mechanisms underlying this activity are mainly cell autonomous and highly pleiotropic. They include a concerted downregulation of 4 key effectors channeling neural SCs to astroglial fates, as well as defective activation of core molecular machineries implementing astroglial differentiation programs. Next, we found that SC Foxg1 levels specifically decline during the neuronogenic-to-gliogenic transition, pointing to a pivotal Foxg1 role in temporal modulation of astrogenesis. Finally, we showed that Foxg1 inhibits astrogenesis from human neocortical precursors, suggesting that this is an evolutionarily ancient trait.

Cortical interlaminar astrocytes across the therian mammal radiation.

Interlaminar astrocytes (ILA) in the cerebral cortex possess a soma in layer I and extend an interlaminar process that runs perpendicular to the pia into deeper cortical layers. We examined cerebral cortex from 46 species that encompassed most orders of therian mammalians, including 22 primate species. We described two distinct cell types with interlaminar processes that have been referred to as ILA, that we termed pial ILA and supial ILA. ILA subtypes differ in somatic morphology, position in layer I, and presence across species. We further described rudimentary ILA that have short GFAP+ processes that do not exit layer I, and "typical" ILA with longer GFAP+ processes that exit layer I. Pial ILA were present in all mammalian species analyzed, with typical ILA observed in Primates, Scandentia, Chiroptera, Carnivora, Artiodactyla, Hyracoidea, and Proboscidea. Subpial ILA were absent in Marsupialia, and typical subpial ILA were only found in Primate. We focused on the properties of pial ILA by investigating the molecular properties of pial ILA and confirming their astrocytic nature. We found that while the density of pial ILA somata only varied slightly, the complexity of ILA processes varied greatly across species. Primates, specifically bonobo, chimpanzee, orangutan, and human, exhibited pial ILA with the highest complexity. We showed that interlaminar processes contact neurons, pia, and capillaries, suggesting a potential role for ILA in the blood-brain barrier and facilitating communication among cortical neurons, astrocytes, capillaries, meninges, and cerebrospinal fluid.

Hypokalemia during antibiotic treatment for bone and joint infections.

PURPOSE: During treatment of bone and joint infections (BJIs) with multiple antibiotic therapy, hypokalemia has been reported as a rare side effect. The aim of this study was to evaluate incidence and risk factors for hypokalemia in a cohort of patients treated with multidrug therapy for BJIs, in a single center. METHODS: We retrospectively reviewed 331 clinical files of 150 consecutive patients (65% males; median age 59 years, 95% CI 55-62) admitted repeatedly to our Osteomyelitis Department for treatment of chronic BJIs. Besides surgical debridement, patients received a combination of oral and intravenous antibiotics. Routine laboratory tests were performed at admittance and repeated at least weekly. Possible hypokalemia risk factors were recorded and analyzed. RESULTS: Progressive kalemia reduction occurred in > 39% of patients during hospitalization; prevalence of marked hypokalemia (K + < 3.5 mEq/l) increased from 5% at admission to 11% (up to 22%) at day 14. Correlated factors were: age ≥ 68 years (p = 0.033), low serum albumin (p = 0.034), treatment with vancomycin (p < 0.001), rifampicin (p = 0.017) and ciprofloxacin (p < 0.001) and use of thiazide (p = 0.007) or loop diuretics (p = 0.029 for K + < 3.5 mEq/l). At multivariate regression analysis, the main determinants of hypokalemia were simultaneous use of diuretics (p = 0.007) and older age (p < 0.049). CONCLUSIONS: Appearance of severe hypokalemia is a frequent event among patients treated for BJIs with multiple antibiotic therapy, when this is prescribed in older age patients and associated with simultaneous use of diuretics. Due to possible increase in mortality risk in the short term, particular caution should be paid during intensive antibiotic treatment in these groups of patients.

Emx2 as a novel tool to suppress glioblastoma.

Glioblastoma is a devastating CNS tumour for which no cure is presently available. We wondered if manipulation of Emx2, which normally antagonizes cortico-cerebral astrogenesis by inhibiting proliferation of astrocyte progenitors, may be employed to counteract it. We found that Emx2 overexpression induced the collapse of seven out of seven in vitro tested glioblastoma cell lines. Moreover, it suppressed four out of four of these lines in vivo. As proven by dedicated rescue assays, the antioncogenic activity of Emx2 originated from its impact on at least six metabolic nodes, which accounts for the robustness of its effect. Finally, in two out of two tested lines, the tumor culture collapse was also achieved when Emx2 was driven by a neural stem cell-specific promoter, likely active within tumor-initiating cells. All that points to Emx2 as a novel, promising tool for therapy of glioblastoma and prevention of its recurrencies.

Emx2 expression levels in NSCs modulate astrogenesis rates by regulating EgfR and Fgf9.

Generation of astrocytes within the developing cerebral cortex is a tightly regulated process, initiating at low level in the middle of neuronogenesis and peaking up after its completion. Astrocytic outputs depend on two primary factors: progression of multipotent precursors toward the astroglial lineage and sizing of the astrogenic proliferating pool. The aim of this study was to investigate the role of the Emx2 homeobox gene in the latter process. We addressed this issue by combined gain- and loss-of-function methods, in vivo as well as in primary cultures of cortico-cerebral precursors. We found that Emx2 overexpression in cortico-cerebral stem cells shrinked the proliferating astrogenic pool, resulting in a severe reduction of the astroglial outcome. We showed that this was caused by EgfR and Fgf9 downregulation and that both phenomena originated from exaggerated Bmp signaling and Sox2 repression. Finally, we provided evidence that in vivo temporal progression of Emx2 levels in cortico-cerebral multipotent precursors contributes to confine the bulk of astrogenesis to postnatal life. Emx2 regulation of astrogenesis adds to a number of earlier developmental processes mastered by this gene. It points to Emx2 as a new promising tool for controlling reactive astrogliosis and optimizing cell-based designs for brain repair.