Prenatal and Postnatal Neurogenesis PDF

“PHYSIOLOGY (Module 2/3)” Prof. Elisabetta Ciani AA 2023-2024 (1 CFU) Neurogenesis and Brain Development Prenatal and postnatal neurogenesis Modulation of postnatal neurogenesis Practical Laboratory Exercitation The human brain is the...

“PHYSIOLOGY (Module 2/3)” Prof. Elisabetta Ciani AA 2023-2024 (1 CFU) Neurogenesis and Brain Development Prenatal and postnatal neurogenesis Modulation of postnatal neurogenesis Practical Laboratory Exercitation The human brain is the most complex known structure in the universe  100 billion neurons each neuron can make connections with more than 1,000 other neurons 60 trillion neuronal connections Can our brain understand how the human brain works?  Neurogenesis Process through which new neurons are generated Neurogenesis includes, but does not end in, the proliferative step: it is a process that begins with the asymmetric division of the precursor and ends only once the new neuron is completely differentiated, integrated, and is able to survive and perform its own functions.  Neurogenesis Two moments of neurogenesis can be distinguished: Developmental neurogenesis, which gives rise to neurons and glial cells that are designed to form the tissues of the nervous system, Adult neurogenesis, connected with the functional plasticity of determined cerebral areas. Neuronal and glial cells are the progeny of neural stem cells.  Stem cells The classical definition of a stem cell is that it possesses two properties: Self-renewal - the ability to go through numerous cycles of cell division while maintaining the undifferentiated state. Potency - the capacity to differentiate into specialized cell types.  What are stem cells? stem cell SELF-RENEWAL POTENCY (unlimited replication) (specialization) specialized cell Stem cell (e.g., muscle, nerve cells) Asymmetric cell division  Where are the stem cells located? Tissue stem cells Embryonic stem cells (Adult stem cells) blastocyst - a very early fetus, child and throughout life stage of development of the embryo 4th to the 14th day after fertilization  Types of stem cells: 1) Embryonic stem cells  Embryonic stem (ES) cells are pluripotent and derive from the internal cell mass of the blastocyst trophoblast blastocele embryo germ layers Ectoderm Mesoderm Endoderm ES are able to differentiate into any of the three germ layers (and form more than 220 cell types)  Embryonic stem cells (ES): Where are they? blastocyst Internal cells = "Internal cellular mass" Culture medium Grown in culture Embryonic stem cells to obtain taken from many more cells internal cell mass External layer of cells = "Trophoderma"  Embryonic stem cells: The challenges skin neurons Embryonic stem cells PLURIPOTENT blood liver  Therapeutic applications of embryonic stem cells Regenerative Medicine Difficult injuries / burns Peripheral Vascular Disease Heart attack Arthritis Parkinson Alzheimer's Diabetes Blood dysfunctions Marrow lesions Tissue Regeneration  Problems in the use of embryonic stem cells  Difficulty in controlling in vitro differentiation  Heteromologous use: possibility of immunological response  Carcinogenicity  Bioethical problems  Types of stem cells: 2) Tissue stem cells (adult stem cells)  Tissue stem cells: Where are they located? eye surface brain skin breast Adipose tissue testicles intestine bone marrow muscles  Tissue stem cells: What can they became? Blood stem cell differentiation found in the bone marrow Only specialized blood cells: Red blood cells, white blood cells, MULTIPOTENT platelets  Neuronal stem cells Progenitors Unipotency or Oligopotency: the ability to differentiate into a single cell type  Types of stem cells: 3) Induced pluripotent stem cells (iPS) Nobel prize in physiology or medicine 2012 John B. Gurdon Shinya Yamanaka  DOGMA: SPECIALIZATION OF CELLS IS NOT REVERSIBLE In a stem cell, DNA (gold) is wrapped loosely around histone proteins (blue). In a differentiated cell, segments of DNA that are not required for the cell's specialized function are shut down and wrapped tightly around histone proteins. John B. Gurdon discovered in 1962 that the specialization of cells is reversible. In a classic experiment, he replaced the immature cell nucleus in an egg cell of a frog with the nucleus from a mature intestinal cell. This modified egg cell developed into a normal tadpole. The DNA of the mature cell still had all the information needed to develop all cells in the frog. John B. Gurdon Dolly (5 July 1996 – 14 February 2003) was a female domestic sheep, and the first mammal cloned from an adult somatic cell, using the process of nuclear transfer.  Shinya Yamanaka discovered more than 40 years later, in 2006, how intact mature cells in mice could be reprogrammed to become immature stem cells. Surprisingly, by introducing only a few genes, he could reprogram mature cells to become pluripotent stem cells, i.e. immature cells that are able to develop into all types of cells in the body. Shinya Yamanaka  Induced pluripotent stem cells (iPS) "Genetic reprogramming" = with transcription regulators: Oct-3/4, Sox2, c-Myc, e Klf4 Body cell Induced pluripotent stem cell (iPS) behaves like an embryonic stem cell differentiation growing iPS cells in culture Advantage: no embryos are necessary All possible types (no ethical problems), no rejection of specialized cells problems.  Future challenges of induced pluripotent stem cells Cell transplantation therapy Disease modelling and drug screening A phase contrast image of human iPSCs maintained in chemical defined medium iPSc Adult stem cells Gage F, SCIENCE 287, 1433, 2000  BRAIN DEVELOPMENT Developmental neurogenesis The brain is formed during the embryonic development of the neural tube, an early embryonic structure. The most anterior part of the neural tube, called the telencephalon, expands rapidly by cell proliferation and gives rise to the brain. Gradually some of the cells stop dividing and differentiate into neurons or glial cells, the main cellular components of the brain. The newly generated neurons migrate to different parts of the developing brain and organize themselves into different brain structures.  Stages of brain development Neural plate formation Neural proliferation Migration and Aggregation Axonal growth and synapse formation Cell death Synaptic rearrangement  During early embryonic development the ectoderm becomes specified to give rise to the epidermis (skin) and the neural plate. neurulation embryo germ layers The neural plate folds outwards during the third week of gestation to form the neural groove.  The neural folds of this groove close to create the neural tube. At the end of the fourth week of gestation E25-E27, the open ends of the neural neurulation tube (neuropores) close. Stages of brain development Neural plate formation Neural proliferation Migration and Aggregation Axonal growth and synapse formation Cell death Synaptic rearrangement  Neuronal proliferation Starts with the closure of the neural tube 6 week (E42)  Up to about E42 in humans, the population of neural progenitor cells divides with a "symmetric" mode of cell division. Symmetric cell division produces two identical neural progenitor cells. Symmetric cell division creates a pool of neural progenitors. 6 week (E42)  Starting from E42, the cell division mode begins to shift from symmetrical to asymmetrical. During asymmetric cell division, two different types of cells are produced. From neural progenitors, asymmetric cell division produces a neuronal progenitor and an immature neuron (neuroblast). The precursors multiply very rapidly at a rate of about 4,000 per second and, at the end of the 16th week, there are more than 20 billion. Two germinal regions, the ventricular zone (VZ) and the subventricular zone (SVZ), generate most of the neurons and glial cells of the mammalian central nervous system. In both humans and rodents (and probably in all mammals) a mitotically active residue of SVZ persists into adulthood. VZ S.L. ANDERSEN; Neuroscience and Biobehavioural Reviews 27 (2003) 3-18 Stages of brain development Neural plate formation Neural proliferation Migration and Aggregation Axonal growth and synapse formation Cell death Synaptic rearrangement Migration As development progresses, the progenitors of VZ become: postmitotic neuroblasts that leave VZ and migrate to the pial surface using the radial glia as their guide. The radial glia, with a cell body located in the VZ and a long extension extending up to the pial surface, favors the migration towards the pial of new neurons. The cells that migrate are immature and without dendrites. Stages of brain development Neural plate formation Neural proliferation Migration and Aggregation Axonal and dendritic growth and synapse formation Cell death Synaptic rearrangement  Axon Growth/Synaptogenesis Once the migration is complete and the structures have formed (aggregation), the axons and dendrites begin to grow to their "mature" form. Axons (with growth cones) and dendrites form synapses with other neurons or target tissues (eg muscle tissue). Growth cones and chemotactic factors are fundamental to this process.  Classes of axon guidance molecules and their receptors Netrins: are secreted molecules that can act to attract or repel axons by binding to their receptors. Slits : Secreted proteins that normally repel growth cones by engaging Robo class receptors. Ephrins: are cell surface molecules that activate Eph receptors on the surface of other cells. This interaction can be attractive or repulsive. Semaphorins: The many types of Semaphorins are primarily axonal repellents, and activate complexes of cell-surface receptors called Plexins and Neuropilins. Cell adhesion molecules (CAMs): Integral membrane proteins mediating adhesion between growing axons and eliciting intracellular signalling within the growth cone. CAMs are the major class of proteins mediating correct axonal navigation of axons growing on axons (fasciculation). Developmental morphogens, such as BMPs, Wnts, Hedgehog, and FGFs Growth factors like NGF Neurotransmitters and modulators like GABA Stages of brain development Neural plate formation Neural proliferation Migration and Aggregation Axonal growth and synapse formation Cell death Synaptic rearrangement  Neuronal Death Between 40-75% of the neurons produced will die after migration (from the 6th month). Neurons die due to failure in the competition for chemoatrattors provided by the target cells. Neurotrophins  Promoting Growth and Survival  Axon guide  Stimulating synaptogenesis  Neuronal Death Release and Neurons receiving Axonal processes uptake of insufficient complete for neurotrophic neurotropic factor die limited factors neurotrophic factor Stages of brain development Neural plate formation Neural proliferation Migration and Aggregation Axonal growth and synapse formation Cell death Synaptic rearrangement Myelination  Synaptogenesis Release and Neurons receiving Axonal processes uptake of insufficient complete for neurotrophic neurotropic factor die limited factors neurotrophic factor  Synaptogenesis & Pruning  In the cortex, the synapses begin to form after neuronal migration, at the 23rd prenatal week.  However, most synapses are formed after birth.  Many are formed randomly (when axons and dendrites meet).  The synapses bloom, then they are selectively pruned.  Up to 100,000 synapses are pruned per second (Kolb, 1999).  Pruning Process Newborns start with about 100 billion neurons and about 50 billion synapses. At three years, the number of synapses has increased twenty times to 1,000 trillion. At puberty the "pruning" process has simplified neural networks to around 500 trillion connections. This pruning is not a random process. Synapses that have been used repeatedly tend to remain. Those that have not been used often enough are eliminated. (Early experiences create brain architecture for life). Brain development is truly a “use it or lose it” process  Synaptogenesis Flourishes up to 2-3 yrs. Synaptic pruning Childhood, up to adult.  Human 6 Years 14 Years Brain at Birth Old Old Golgi method or chrome-silver impregnation. Golgi's method stains a limited number of cells at random in their entirety.  Golgi's method Golgi's method is a silver staining technique that is used to visualize nervous tissue under light microscopy. The method was discovered by Camillo Golgi, an Italian physician and scientist, who published the first picture made with the technique in 1873. It was initially named the black reaction by Golgi, but it became better known as the Golgi stain or later, Golgi method.  A lesson from Fragile-X syndrome Fragile X syndrome is a genetic condition that causes a range of developmental problems including learning disabilities and cognitive impairment. It is the most common hereditary form of mental retardation after Down Syndrome. The disease is caused by the mutation of the FMR1 gene (Fragile X Mental Retardation-1) located on the long arm of the X chromosome. Usually, males are more severely affected by this disorder than females. The DNA mutation modifies the structure of the X chromosome "choke" in the terminal region (Xq27.3), where the FMR1 gene "X-FRAGILE" is located.  A lesson from Fragile-X syndrome A defective FMR1 (fragile X-mental retardation protein) gene suppresses production of proteins that stimulate pruning Excess synapses not pruned sufficiently “Noise” in the neural system causes MR LESS IS MORE! Pruning is important. » Greenough & Black, 1999; Nelson, de Haan, Thomas, 2006 Neurons in brains from people with autism do not undergo normal pruning during childhood and adolescence. The images show representative neurons from a unaffected brain (left) and brain from an autistic patient (right); the spines on the neurons indicate the location of synapses. (Image: Guomei Tang and Mark S. Sonders/CUMC)  Pruning During childhood, pruning causes a loss of up to 10% of the gray matter volume in the cortex (with 607% contraction of the frontal lobes between 13 and 18 years of age). The weight of the human brain is, however, maintained due to an increase in myelination (Huttenlocher, 1999)  White Matter Growth Associated with Post-natal Proliferation of Oligodendrocytes and Myelin Deposition 2 1 Cerebral WM 0 -1 -2 -3 0 10 20 30 40 50 60 70 80 90 100 Age Postnatal Neurogenesis  The Problem of Adult Neurogenesis The great Spanish neuroscientist Santiago Ramón y Cajal (1852-1934), devoted himself to the study of damaged brain regeneration. To his great disappointment, he couldn't find any evidence of Golgi and Cajal shared the Nobel proliferating neurons in the adult brain. Prize in 1906 for their studies on the nervous system This gave rise to the Dogma: Neurogenesis does NOT exist in adults  POSTNATAL NEUROGENESIS As demonstrated by the pioneering studies of Joseph Altman (1965) and, subsequently, of Fred H. Gage (1992), in the adult mammalian brain neurogenesis remains at the level of the subgranular zone of the dentate gyrus (SGZ) of the hippocampus and subventricular zone of the lateral ventricles (SVZ). In fact, the presence of neuronal stem cells has been demonstrated in these areas.  METHODS OF STUDYING NEUROGENESIS To study adult neurogenesis in vivo, it is necessary to identify the newly generated neurons among billions of pre-existing neurons in the adult brain. There are three main approaches used to label newborn neurons: 1- Incorporation of exogenous nucleotide analogues. 2- Genetic marking with retroviruses that infect only dividing cells. 3- Labeling of endogenous proliferation markers.  Injection of exogenous nucleotides Tritiated thymine (3HThy) followed by autoradiographic detection. 5’-Bromo-2-deoxyuridine (BrdU) followed by immunohistochemical detection. One useful feature of BrdU is its long-term retention in divided cells and its passage to their daughter cells. This feature can be used to trace the cell lineage and cell survival. 3HThy autoradiographic detection by a photographic emulsion  BrdU BrdU B A A G G C B C C T T G A A T IMMUNOISTOCHIMICA  VIRAL VECTOR In addition to thymidine analogues, retroviruses have also been used to introduce markers into replicating cells. The virus is usually a modified nonreplicative oncoretrovirus (the genes required for replication are deleted) Local infusion of the retrovirus into the brain region of interest. Retroviruses must integrate into the genome, and can do so only when the cell divides. Viral Vector virus Retroviral vectors drive expression of green fluorescent protein (GFP).  Jellyfish Aequorea victoria If GFP is excited by radiation at a specific wavelength, it is able to re-emit bright green light (fluorescence properties). Present on the west coast of North America. Same litter  Endogenous markers Ki-67, a nuclear protein expressed in all phases of the cell cycle except the resting phase (G1, S, G2 and M phases, but not in G0) PCNA (Proliferating Cell Nuclear Antigen), an auxiliary protein of DNA polymerase-delta. PCNA) is elevated in the nucleus during late G1 phase immediately before the onset of DNA synthesis, becoming maximal during S-phase and declining during G2 and M phases. Ki-67 BrdU Merge  CELL FATE The fate of BrdU-positive cells can be determined with double (triple) immunofluorescent labeling for BrdU and cell-specific markers. GFAP (glial fibrillay acidic protein) is a marker of glial cells (astroglia). NeuN (neuronal specific nuclear protein) is a marker of mature neurons..  FATE BrdU+NeuN BrdU+GFAP  POSTNATAL NEUROGENESIS The two main neurogenic areas of the adult mammalian brain are: the subventricular zone (SVZ) of the lateral ventricles, which generates the neurons of the olfactory bulbs, the sub-granular zone (SGZ) of the DG of the hippocampus responsible for the production of neurons in the granular layer of the dentate gyrus (DG) (Taupin and Gage, 2002). In rodents SVZ produces 30,000 neurons / day SGZ produces 9,000 neurons / day https://en.wikipedia.org/wiki/Lateral_ventricles  THE STRUCTURE OF THE SVZ The human and rodent SVZ is a region that lies immediately beneath the ependymal layer on the lateral wall of the lateral ventricles; thus, it is a niche region that is in close proximity to the nutrients and growth factors that are present in the cerebrospinal fluid (CSF) of the ventricles.  SVZ Neurogenic Niche The three-dimensional composition and organization of the murine SVZ, has been studied, ultrastructurally and immunohistochemically, by Doetsch and collaborators (1997) who identified the presence of three cell types: astrocytes or cells of type B1 and B2, the cells of type C (mitotically active immature precursors) and neuroblasts or type A cells. Type A cells are small, when colored they appear dark and correspond to neuroblasts. Type B1 and B2 cells share characteristics similar to astrocytes. Type B1 cells are immature astrocytes, while type B2 are probably neural stem cells. Type C cells are larger with slightly stained nuclei. They are highly mitotic and therefore represent transient amplification progenitor cells "transient amplifying cells". Neuroblasts (Type A) migrate to the olfactory bulb along a migration path known as "rostral migratory stream" (RMS). More than 30,000 neuroblasts in rodents exit SVZ every day and enter the RMS, where they migrate in chains through the tubular structures formed by specialized astrocytes. The SVZ neuroblasts, after having migrated along the RSM up to the olfactory bulb, mature in olfactory inhibitor interneurons (granule cells and periglomerular cells). Both types of cells make local contacts in the bulb, modulating processing of sensory information from the olfactory projection neurons, the mitral and plume cells. olfactory mucosa  HIPPOCAMPAL CIRCUIT ventromedial region of each temporal lobe Giulio Cesare Aranzio (1585) https://it.wikipedia.org/wiki/Ippocampo_(anatomia)#/media/File:Hippocampus.gif https://en.wikipedia.org/wiki/Hippocampus#/media/File:Hippocampus_and_seahorse_cropped.JPG  HIPPOCAMPAL CIRCUIT ventromedial region of each temporal lobe  Classification of Memory Three stages of memory 1 sec 20-45 sec days, weeks, years… By classifying the memory in terms of "type of information" we can distinguish two types of memory that concern practical skills and knowledge respectively 1. IMPLICIT or PROCEDURAL MEMORY (non-declarative memory): a memory that concerns the modalities of execution of some act and that is recalled to the mind in an unconscious way. It is a memory connected with the training for the execution of motor or perceptive tasks, of a reflex type. 2. EXPLICIT MEMORY (declarative memory): knowledge of facts relating to persons, places, things and their meaning. It is a memory that is called to mind with deliberate and conscious efforts. The hippocampus is involved in:  the consolidation of short-term memory  long-term declarative (explicit) memory  the spatial memory  Tests for spatial memory Morris Water Maze Barnes Test The SGZ of the hippocampus gives rise to progenitors of the dentate gyrus. SGZ stem cells give rise to intermediate progenitors leading to the production of ~ 9,000 new cells per day in young rodents. These progenitors mature locally in granular neurons of the dentate gyrus and send axonal projections to the CA3 area of the hippocampus. Postmortem tissue from the hippocampus and the subventricular zone of caudate nucleus was obtained from cancer patients (n = 5) who received one intravenous infusion of bromodeoxyuridine (BrdU) for diagnostic purposes. This study was exceptionally important in that it provided strong evidence for the presence of adult neurogenesis in humans However, it did not enable any quantitative estimate -does adult neurogenesis decrease with primate evolution?? -is the extent of this process in humans sufficient to have any functional impact?? In order to be able to study cell turnover dynamics in humans, a strategy to retrospectively birth date neogenerated cells has been developed.  This strategy takes advantage of the elevated atmospheric 14C levels caused by above ground nuclear bomb testing 1955–63 during the Cold War CO2 + H2O + photons C6H12O6 + O2 + H2O When a cell divides and duplicates its genome, it will integrate 14C in DNA with a concentration corresponding to that in the atmosphere at any given time. Measuring the 14C concentration in genomic DNA allows determination of when cells were born. One-third of hippocampal neurons are subject to exchange The annual turnover rate is 1.75% within the renewing fraction in adult humans (~700 new neurons per day in each hippocampus)  The extent of adult neurogenesis is comparable in middle-aged humans and mice Interspecies comparison of the extent of adult hippocampal neurogenesis. Representation of the proportion of newborn neurons to old neurons in the dentate gyrus  What are these new cells doing? What are the thousands of new cells generated each day doing? What are they responsible for? Are they important for memory?  FUNCTIONAL MEANING OF ADULT NEUROGENESIS The functional significance of adult neurogenesis is uncertain. It is however proven that: - the destruction of hippocampal stem cells (following irradiation) produces a reduction in behavioral performance dependent on this area, as evidenced in spatial memory tasks. - stimuli that increase neurogenesis enhance hippocampal performance. Perturbations in normal neurogenesis have been associated with a number of diseases, such as depression and epilepsy.  Postnatally-forming precursors of neurons in the hippocampus are highly vulnerable and are killed with low-level X- irradiation. This permanently interferes with adult neurogenesis.  Hippocampal X-irradiation: Behavioral Studies SPONTANEOUS ALTERNATION TEST. Rat was given 8 Will a rat re-enter an arm doses of X-rays of a T-maze that it has between P2 and already explored? P15 and behavioral tested at P60. Normal rats spontaneously alternate above chance level on the second trial.  Hippocampal X-irradiation: Behavioral Studies The X-irradiated animals have SHORT-TERM MEMORY DEFICITS since they readily re-enter the same arm of a T- maze in successive trials. Hippocampal X-irradiation: Behavioral Studies The PASSIVE AVOIDANCE apparatus first shapes hungry rats to eat from a food cup when the door is opened. A day later the rats are shocked when they eat from the food cup. On the next testing day, the hungry rats are again placed in the apparatus and the door is opened. The time it takes for the rat to approach the food cup is recorded. Normal rats stay in the start box for a long time to passively avoid being shocked.  Hippocampal X-irradiation: Behavioral Studies The X-irradiated animals have LEARNING DEFICITS to avoid shock because they approach the food cup after a shorter delay than normal rats.  CONCLUSION: The “Good” News This research, and that of many other developmental neurobiologists, has established that the regenerative capacity of the nervous system is FAR GREATER than was believed. Is it possible that adult-generated neurons can be coaxed into therapies to effectively remedy developmental disorders like autism, or degenerative disorders like Alzheimer’s disease?  CONCLUSION: The “Bad” News The stem cells that give rise to neurons in adult brains are highly vulnerable and can be killed by exposure to radiation, alcohol, drugs, etc. The absence of postnatal and adult-generated neurons affects the function of specific brain structures and leads to learning disabilities, abnormal behavior, and other disorders.  The discovery that neurogenesis persists even into adulthood has launched enormous efforts to: - characterize how the new neurons differentiate and integrate into adult neuronal circuitry, - understand the consequences of a lack of neurogenesis in neuropsychiatric and pathological processes, - analyze whether endogenous neuronal stem cells can be exploited to repair the brain However, we are only beginning to understand the cellular and molecular mechanisms that regulate the process of neurogenesis in the adult brain, and how this can contribute to neurological diseases. 1996