Pasko Rakic

Reduced Apoptosis and Cytochrome c–Mediated Caspase Activation in Mice Lacking Caspase 9

Caspases are essential components of the mammalian cell death machinery. Here we test the hypothesis that Caspase 9 (Casp9) is a critical upstream activator of caspases through gene targeting in mice. The majority of Casp9 knockout mice die perinatally with a markedly enlarged and malformed cerebrum caused by reduced apoptosis during brain development. Casp9 deletion prevents activation of Casp3 in embryonic brains in vivo, and Casp9-deficient thymocytes show resistance to a subset of apoptotic stimuli, including absence of Casp3-like cleavage and delayed DNA fragmentation. Moreover, the cytochrome c-mediated cleavage of Casp3 is absent in the cytosolic extracts of Casp9-deficient cells but is restored after addition of in vitro-translated Casp9. Together, these results indicate that Casp9 is a critical upstream activator of the caspase cascade in vivo.

Absence of excitotoxicity-induced apoptosis in the hippocampus of mice lacking the Jnk3 gene

Excitatory amino acids induce both acute membrane depolarization and latent cellular toxicity, which often leads to apoptosis in many neurological disorders,. Recent studies indicate that glutamate toxicity may involve the c-Jun amino-terminal kinase (JNK) group of mitogen-activated protein (MAP) kinases. One member of the JNK family, Jnk3, may be required for stress-induced neuronal apoptosis, as it is selectively expressed in the nervous system,. Here we report that disruption of the gene encoding Jnk3 in mice caused the mice to be resistant to the excitotoxic glutamate-receptor agonist kainic acid: they showed a reduction in seizure activity and hippocampal neuron apoptosis was prevented. Although application of kainic acid imposed the same level of noxious stress, the phosphorylation of c-Jun and the transcriptional activity of the AP-1 transcription factor complex were markedly reduced in the mutant mice. These data indicate that the observed neuroprotection is due to the extinction of a Jnk3-mediated signalling pathway, which is animportant component in the pathogenesis of glutamate neurotoxicity.

Neuronal migration, with special reference to developing human brain: a review

A general rule in the developing central nervous system is that cells are generated in sites different from those in which they will later reside. The intervening migrations, particularly in the human nervous system, form the subject of this review. The basic columnar organization in the early stages of development favors radial migration of cells. During later stages in primates, when young neurons migrate to the distant cerebral cortex, they follow radial glial guides across the widening intermediate zone as they pass from the juxtaventricular site of genesis to the cortical plate. Somas of later-generated cells take positions external to somas of their predecessors. The final position along the radial vector may be influenced by afferent axons. Cell relationships in the developing cerebellar cortex are essentially similar, though the key migration of granule cell neurons is in the reverse direction, from the external surface inward past Purkinje dendrites and somas. Bergmann glial fibers provide the radial guidance in this instance. The degree of dependence of developing neurons upon other cells and cell processes in their immediate environment has been clarified by study of mutant mice in which cerebral or cerebellar cortices are malformed. Other special migrations in the fetal human brain are reviewed, particularly the passage of neurons from the rhombic lip through the transient corpus pontobulbare to mainly the inferior olives and pontine gray nuclei, and from the ganglionic eminence of the cerebrum through the corpus gangliothalamicum into the pulvinar region of the thalamus. It was suggested that the special relationships involved in these various migrations are probably mediated by cell surface properties, and that such surface properties will come to be defined through analysis of reaggregation tissue cultures, experimental and natural chimeras, and by immunological definition of antigens on CNS cells at different stages of development.

The Jnk1 and Jnk2 protein kinases are required for regional specific apoptosis during early brain development.

The c-Jun NH2-terminal kinase (Jnk) family is implicated in apoptosis, but its function in brain development is unclear. Here, we address this issue using mutant mice lacking different members of the family (Jnk1, Jnk2, and Jnk3). Mice deficient in Jnk1, Jnk2, Jnk3, and Jnk1/Jnk3 or Jnk2/Jnk3 double mutants all survived normally. Compound mutants lacking Jnk1 and Jnk2 genes were embryonic lethal and had severe dysregulation of apoptosis in brain. Specifically, there was a reduction of cell death in the lateral edges of hindbrain prior to neural tube closure. In contrast, increased apoptosis and caspase activation were found in the mutant forebrain, leading to precocious degeneration. These results suggest that Jnk1 and Jnk2 regulate region-specific apoptosis during early brain development.

A small step for the cell, a giant leap for mankind: a hypothesis of neocortical expansion during evolution

The more than 1000-fold increase in the cortical surface without a comparable increase in its thickness during mammalian evolution is explained in the context of the radial-unit hypothesis of cortical development. According to the proposed model, cortical expansion is the result of changes in proliferation kinetics that increase the number of radial columnar units without changing the number of neurons within each unit significantly. Thus, mutation of a regulatory gene(s) that controls the timing and ratio of symmetric and asymmetric modes of cell divisions in the proliferative zone, coupled with radial constraints in the distribution of migrating neurons, could create an expanded cortical plate with enhanced capacity for establishing new patterns of connectivity that are validated through natural selection.

Evolution of the neocortex: a perspective from developmental biology.

The enlargement and species-specific elaboration of the cerebral neocortex during evolution holds the secret to the mental abilities of humans; however, the genetic origin and cellular mechanisms that generated the distinct evolutionary advancements are not well understood. This article describes how novelties that make us human may have been introduced during evolution, based on findings in the embryonic cerebral cortex in different mammalian species. The data on the differences in gene expression, new molecular pathways and novel cellular interactions that have led to these evolutionary advances may also provide insight into the pathogenesis and therapies for human-specific neuropsychiatric disorders.

Microarray analysis of microRNA expression in the developing mammalian brain

BackgroundMicroRNAs are a large new class of tiny regulatory RNAs found in nematodes, plants, insects and mammals. MicroRNAs are thought to act as post-transcriptional modulators of gene expression. In invertebrates microRNAs have been implicated as regulators of developmental timing, neuronal differentiation, cell proliferation, programmed cell death and fat metabolism. Little is known about the roles of microRNAs in mammals.ResultsWe isolated 18-26 nucleotide RNAs from developing rat and monkey brains. From the sequences of these RNAs and the sequences of the rat and human genomes we determined which of these small RNAs are likely to have derived from stem-loop precursors typical of microRNAs. Next, we developed a microarray technology suitable for detecting microRNAs and printed a microRNA microarray representing 138 mammalian microRNAs corresponding to the sequences of the microRNAs we cloned as well as to other known microRNAs. We used this microarray to determine the profile of microRNAs expressed in the developing mouse brain. We observed a temporal wave of expression of microRNAs, suggesting that microRNAs play important roles in the development of the mammalian brain.ConclusionWe describe a microarray technology that can be used to analyze the expression of microRNAs and of other small RNAs. MicroRNA microarrays offer a new tool that should facilitate studies of the biological roles of microRNAs. We used this method to determine the microRNA expression profile during mouse brain development and observed a temporal wave of gene expression of sequential classes of microRNAs.

Principles of neural cell migration

A basic property of immature neurons is their ability to change position from the place of their final mitotic division in proliferative centers of the developing brain to the specific positions they will occupy in a given structure of the adult nervous system. Proper acquisition of neuron position, attained through the process of active migration, ultimately affects a cells morphology, synaptic connectivity and function. Although various classes of neurons may use different molecular cues to guide their migration to distant structures, a surface-mediated interaction between neighboring cells is considered essential for all types of migration. Disturbance of this cell-cell interaction may be important in several congenital and/or acquired brain abnormalities. The present article considers the basic mechanisms and principles of neuronal cell migration in the mammalian central nervous system.

A Golgi study of radial glial cells in developing monkey telencephalon: morphogenesis and transformation into astrocytes.

SummaryRadial glial cells (epithelial cells of Ramón y Cajal) impregnated by a modified del Rio Hortega rapid Golgi method were studied in the occipital lobes of 38 rhesus monkeys from embryonic day 48 (E48) to birth which occurs at E165 and in 27 postnatal animals to day 365 (P365). Some radial glial cells are already recognized at E48 by their bipolar shape and elongated radial fiber, which terminates with characteristic endfeet on the walls of blood vessels or at the pial surface. At slightly older ages-between E60 and E70-all cells spanning the cerebral wall develop lamellate expansions along their radial fiber and their endfeet become PAS positive. After E60, some radial glia detach from the ventricular surface and their somas become displaced outwards in the cerebral wall. After this age, radial glial cells are easily distinguished from migrating neurons by their larger oval nucleus located in the ventricular or subventricular zone, radial fiber extending outwards to the pial surface where it terminates in one or more endfeet, and the delicate lamellate expansions on both radial fiber and soma.Displaced radial glial cells have more closely packed lamellate expansions and display a range of transitional shapes leading to either fibrous or protoplasmic astrocytes. Between E95 and E140, when neuron migration to the visual cortex tapers off, perikarya of displaced radial glial cells form a conspicuous band at the outer border of the subventricular zone. Numerous transitional forms are present in the cortical plate at this age. After birth, fewer radial glial fibers are present in occipital lobe and their length is difficult to determine in the convoluted lateral cerebral wall expanded up to 10–20 mm. However, at P7 and P20, many radial fibers still span the medial cerebral wall in the depth of the calcarine fissure where it remains less than 2 mm thick. Even here, no fibers spanning the cerebral wall were seen in 17 animals from P50 to P200 despite the presence of well-impregnated transitional forms situated near the lateral ventricle and myriad astrocytes dispersed throughout the hemisphere. By P365, end of the first year, the few short remaining radial fibers belong to ependymal cells or mature astrocytes while all immature transitional forms have disappeared.

Origin of GABAergic neurons in the human neocortex.

The mammalian neocortex contains two major classes of neurons, projection and local circuit neurons. Projection neurons contain the excitatory neurotransmitter glutamate, while local circuit neurons are inhibitory, containing GABA. The complex function of neocortical circuitry depends on the number and diversity of GABAergic (γ-aminobutyric-acid-releasing) local circuit neurons. Using retroviral labelling in organotypic slice cultures of the embryonic human forebrain, we demonstrate the existence of two distinct lineages of neocortical GABAergic neurons. One lineage expresses Dlx1/2 and Mash1 transcription factors, represents 65% of neocortical GABAergic neurons in humans, and originates from Mash1-expressing progenitors of the neocortical ventricular and subventricular zone of the dorsal forebrain. The second lineage, characterized by the expression of Dlx1/2 but not Mash1, forms around 35% of the GABAergic neurons and originates from the ganglionic eminence of the ventral forebrain. We suggest that modifications in the expression pattern of transcription factors in the forebrain may underlie species-specific programmes for the generation of neocortical local circuit neurons and that distinct lineages of cortical interneurons may be differentially affected in genetic and acquired diseases of the human brain.