Adult Neurogenesis —It's just nice to know it's there!

Neurogenesis in substantia nigra of Parkinsonian brains?

2011-10-14T05:28:00.000-07:00

Parkinson's disease is a neurodegenerative disorder characterized by a progressive loss of dopaminergic neurons in the nigrostriatal projection. Transplantation of fetal dopaminergic precursor cells has paved the way for the possibility of a cell replacement therapy that could ameliorate clinical symptoms in affected patients.

Neural stem cells have been identified in the neurogenic brain regions, where neurogenesis is constitutively ongoing, but also in the non-neurogenic zones, such as the midbrain and the striatum, where neurogenesis is not thought to occur under normal physiological conditions:

A) The ventral midbrain in a rat, stained immunohistochemically for tyrosine hydroxylase (green), comprises the substantia nigra pars compacta (SNc) containing predominantly dopaminergic neurons giving rise to nigrostriatal axons, the substantia nigra pars reticulata (SNr) containing mainly GABAergic neurons and some dopaminergic neurons, and the ventral tegmental area (VTA) containing mainly dopaminergic neurons giving rise to mesolimbic axons.

B) In Parkinson’s disease and corresponding animal models (here exemplified by a 6-OHDA-lesioned rat), a preferential degeneration of the dopaminergic neurons in the substantia nigra with relative sparing of the VTA is observed.

C) In the parenchyma of the normal adult ventral midbrain, presence of neural stem cells (NSCs) has been reported, which unfold their neurogenic potential once explanted and stimulated with appropriate cues in vitro.

D) In vivo, however, the neurogenic potential of the adult parenchymal NSCs in the ventral midbrain appears to be restricted by presently unidentified inhibiting cues (flashes), which might be emitted for example by local glial cells or mature neurons, rendering the substantia nigra in the adult brain a primarily non-neurogenic area.

A detailed understanding of the factors governing adult neural stem cells in vivo may ultimately lead to elegant cell therapies for neurodegenerative disorders such as Parkinson's disease by mobilizing autologous endogenous neural stem cells to replace degenerated neurons.

Neurogenesis and Parkinson’s Disease

2011-10-09T14:54:00.000-07:00

Parkinson’s disease (PD) is the most common neurodegenerative movement disorder, rising in incidence after the age of sixty. Clinically, PD is characterized by progressive motor symptoms (bradykinesia, resting tremor, rigidity, and postural instability), mainly linked to the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc), which leads to depletion of dopamine in the striatum, the main target of the axonal projections arising from the substantia nigra (see figure 1). Despite the availability of effective symptomatic drugs, there is presently no cure for PD, and all attempts to slow the neuronal cell loss in the disease have failed.

Novel cell-based therapies aiming for a stimulation of endogenous dopamine production at a constant rate within the brain might provide a more physiological and elegant way to overcome the dopaminergic deficiency in Parkinsonian brains.

Initial enthusiasm for an experimental cell replacement therapy by grafting fetal neuronal precursor cells into the striatum has vanished after two double-blind placebo-controlled clinical trials showing only moderate symptomatic improvement and the occurrence of severe disabling dyskinesia.

Neural stem cells (NSCs) derived from adult tissues are an attractive alternative source for cell therapy for PD. They overcome the ethical issues inherent to the use of human fetal tissue or embryonic stem cells. NSCs derived from adult tissue also open the possibility for autologous transplantations without immunological complications, where NSCs are taken out from the patient, expanded, differentiated and re-implanted back as dopaminergic precursor cells.

The discovery of constitutive ongoing neurogenesis in the adult human brain has challenged the traditional view of a fixed circuitry in functionally normal brains, and has raised high hopes that the adult brain may have the capacity for self-renewal after injury, thereby avoiding the need for transplantation. Primary neural precursor cells reside in specialized zones called “neurogenic niches”. A population of NSCs preserves enough germinal character to maintain neurogenesis throughout life and, once differentiated, their daughter cells integrate into already existing neuronal networks. Whether adult neurogenesis can be induced under certain circumstances in regions that lack constitutive adult neurogenesis remains controversial, but several studies have reported the isolation of NSCs from different regions of the adult brain, including the SNc.

The clinical motor dysfunction in PD is primarily linked to the depletion of dopamine in the striatum consecutive to the loss of the large dopaminergic neurons in the SNc. The classical view is that this is the result of a progressive degeneration process triggered by unidentified pathogenic factors. This hypothesis was challenged in 2001 by Armstrong and Barker (2001) who proposed the concept that the dopaminergic population in the SNc undergoes a continuous turnover with a low rate of spontaneous neuronal cell loss and an equilibrated rate of constitutive neurogenesis to replace the lost neurons. According to this concept, the reduced number of dopaminergic neurons in the SNc in PD would result from a reduced rate of adult neurogenesis rather than by an increased rate of neuronal cell loss. In 2003, Zhao and coworkers presented experimental findings which suggested that new dopaminergic neurons would indeed be continuously born in the SNc of adult mice to replace spontaneously degenerating neurons. According to this hypothesis, the dopaminergic neuronal population in the SNc would undergo a complete turnover during the lifespan of a mouse. Since then, the hypothesis that the generation of new dopaminergic neurons in the adult SNc could occur spontaneously or as a consequence of lesion-induced repair-mechanisms has received much attention.

Cells expressing polysialylated neural cell adhesion molecule (PSA-NCAM), a sensitive marker of immature neuroblasts, have been detected in the postmortem substantia nigra of human PD patients. This has been put forward as an argument for ongoing neurogenesis in the adult human SNc. However, PSA-NCAM is also expressed in reactive astrocytes and in neurites of mature neurons undergoing plastic changes. Thus, these findings do show that some degree of cellular plasticity is maintained in the SNc, even in aged human PD patients, but they are no strong evidence in favour of neurogenesis in the adult human substantia nigra in PD.

In the adult rodent SNc, the existence of cells expressing the EGF-receptor has been demonstrated, which characterizes rapidly proliferating neural precursor cells (C-Cells) in the subventricular zone (SVZ). Indeed, the adult rodent midbrain appears to contain progenitor cells with the potential to differentiate into astrocytes, oligodendrocytes and neurons, particularly dopaminergic neurons, once these precursor cells are explanted and grown in vitro.

Dopaminergic neurons reside in the substantia nigra, pars compacta (SNc), which is located in the ventral midbrain, and send axonal projections to the striatum, which is situated in the forebrain. Neural stem cells are located in the adult SVZ, immediately adjacent to the striatum, with is rich in dopaminergic afferents from the SNc. The stem cells in the SVZ give rise to neuroblasts, which migrate along the rostral migratory stream (RMS) to the olfactory bulb (OB). In the 6-hydroxydopamine animal model of Parkinson’s disease, the nigral dopaminergic neurons are destroyed unilaterally by means of a stereotactic injection of the toxin, as indicated by the needle. The consecutive depletion of dopamine in the striatum leads to a decreased proliferation of progenitor cells in the SVZ (see figure 2).

However, it remains controversial whether these precursor cells can unfold their neurogenic potential within the parenchyma of the adult brain. There are some reports describing BrdU⁺ neurons with dopaminergic cytoplasm in the substantia nigra of rodents, which occurred either spontaneously or even more pronouncedly after experimentally induced degeneration of the nigrostriatal dopaminergic projection. So far, four articles have reported the existence of in vivo neurogenesis in the adult SNc of experimental animals.

Zhao and co-workers used several methods to provide evidence in favour of an ongoing turnover of the dopaminergic neuronal cell population in the rodent SNc. These authors described a constitutive nigral neurogenesis and, in addition, an increased neurogenesis in response to an MPTP-induced lesion of the nigrostriatal neurons. The authors suggested that the newly generated cells are derived from stem cells lining the cerebroventricular system. Unfortunately, the illustrations presented leave a degree of uncertainty about the newborn nature of the dopaminergic neurons, since the co-localisation of a tyrosine hydroxlyase (TH)⁺ cytoplasm around the BrdU⁺ nucleus is not unequivocally demonstrated in all three dimensions.

Van Kampen and Robertson (2005) reported that on average 10 BrdU⁺ cells can be found in the substantia nigra of healthy adult female rats, of which 15 % co-expressed the neuronal marker NeuN and 8% the catecholaminergic marker TH. These findings imply that a low rate of physiological neurogenesis seems to occur in the rodent SNc. By chronic infusion of 7-OH-DPAT, a D3 receptor agonist, the total number of BrdU⁺ cells was reported to increase to approximately 70 cells on average, of which 30% co-expressed NeuN and 20% TH. The authors concluded that D3 receptor stimulation would induce a strong precursor cell proliferation and would promote the expression of a dopaminergic phenotype in the newborn neurons.

One year later, Van Kampen and Eckman, (2006) published an increase of BrdU/NeuN/TH triple labelled cells also in a 6-OHDA rat model of PD, when animals were treated with 7-OH-DPAT. In contrast to Zhao et al. (2003), Van Kampen and Eckman (2006) proposed a local parenchymal origin of the newborn cells based on the absence of co-labeling with doublecortin, a marker for migrating neuroblasts. In the confocal pictures provided in this publication, the nuclear marker BrdU and the cytoplasmic marker TH are stained in the same cellular compartment, suggesting the rupture of the nuclear membrane, which is a typical feature of apoptotic cell death or antibody ‘bleeding’. Thus, it is uncertain whether the authors have indeed depicted genuinely newborn neurons or perhaps rather pre-existing neurons undergoing a form of mitosis-like apoptosis, which has been demonstrated to occur in the 6-OHDA and MPTP models of PD and in human PD patients. It might therefore be that the increase in number of TH⁺ nigral neurones in 6-OHDA lesioned rats after 7-OH-DPAT treatment, reported by Van Kampen and Eckman, does not represent neurogenesis, but rather other mechanisms of cellular plasticity such as phenotypic recovery or phenotype shifting.

Shan and co-workers reported findings suggestive of constitutive levels of neurogenesis, in particular dopaminergic neurogenesis, in mice. This phenomenon was reported to increase after MPTP-induced dopaminergic depletion. In their experiments, the authors used brain slices with 12µm diameter, which makes a correct interpretation of their findings impossible, since the nigral cell diameter can be up to 15µm. Thus, according to the rules of stereology, a newborn dopaminergic neuron with a BrdU⁺ nucleus can by no means be distinguished from a BrdU⁺ nucleus of an astrocytic satellite cell invaginated into a pre-existing dopaminergic neuron. Besides, in the pictures shown, the BrdU staining is stronger in the cytoplasm than in the nucleus, suggesting that the BrdU incorporated into the nuclear DNA reflects an incorporation of a small number of BrdU molecules into the DNA strand as occurs during DNA repair rather than a comprehensive DNA duplication during mitotic cell division.

Furthermore, several independent groups have tried unsuccessfully to obtain evidence of generation of BrdU⁺ dopaminergic neurons in the adult rodents’ substantia nigra in an unlesioned condition.

Even the additional exposure to factors known to increase neurogenesis in the SVZ or the SGZ, such as transforming growth factor-alpha (TGFα), glia-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), platelet-derived growth factor-BB (PDGF-BB), liver growth factor (LGF), or housing in an enriched environment and physical activity, did not lead to a detectable rate of neurogenesis in the SNc in a in vivo condition. It has been reported that an increase in the number of dopaminergic nigral neurones can be induced by the administration of GDNF. This effect, however, resulted from an upregulation of dopaminergic neuronal markers in already existing neurons rather than from the generation of new neurones.

The Subventricular Zone

2011-10-04T06:21:00.000-07:00

The subventricular zone (SVZ) contains the largest pool of NSCs in the adult mammalian brain, including humans.

In rodents, the adult SVZ contains four distinct cell types defined by morphological, molecular markers, and electrophysiological properties: 1) neural progenitor (type A) cells express PSA-NCAM, Tuj1, and Hu; 2) NSCs in the SVZ are protoplasmic astrocytes (type B cells) and express nestin, vimentin and GFAP; 3) transit amplifying (type C) cells are nestin positive and form clusters adjacent to the chains of migrating neuroblasts throughout the SVZ; 4) ependymal ciliated (type E) cells separate the SVZ from the ventricular cavity (see figure).

The organization of the adult human SVZ is significantly different from that of rodents. In adult rodents, SVZ astrocytes (Type B cells) are located next to the ependymal layer. In contrast, in the adult human brain SVZ astrocytes are not found adjacent to the ependymal cells and no chains of migrating neuroblasts are found in this region. Instead, the cell bodies of human SVZ astrocytes accumulate in a band or ribbon separated from the ependymal layer by a gap that is largely devoid of cells (see figure).

Neural progenitors migrate from the SVZ through the rostral migratory stream (RMS), and after reaching the olfactory bulb (OB) they originate olfactory granule and periglomerular interneurons. Granule neurons are all GABAergic, whereas only 40% of the periglomerular neurons are GABAergic and, of those, 65% are also dopaminergic. Under normal conditions, neural progenitors in the SVZ remain as non-differentiated non-excitable cells. They begin to display properties of mature interneurons only on their arrival to the olfactory bulb. However, in humans, the extent of SVZ neurogenesis and the existence of the RMS are controversial.

The existence of functional adult neurogenesis in the SVZ raises the exciting possibility that manipulating endogenous neural progenitors may lead to successful cell replacement therapies for various degenerative neurological diseases, including Parkinson disease. Infusion of mitogens, growth factors or chemokines into the striatal parenchyma may activate the proliferation of neural stem cells in the SVZ and the migration of their progeny into the striatum to differentiate into dopaminergic neurons to replace the function of the degenerating nigrostriatal dopaminergic afferents.

Adult Neurogenesis

2011-10-01T17:55:00.000-07:00

The dogma that the adult mammalian brain is incapable of generating new neurons has been finally overcome, since neurogenesis in the adult brain was described in the subventricular zone (SVZ) adjacent to the lateral ventricles and in the subgranular zone (SGZ) in the dentate gyrus of the hippocampus (see figure).

In contrast to these two so-called neurogenic areas, the remainder of the brain is considered to be non-neurogenic, implying that no new neurons are produced there under normal conditions. This distinction seems critical, since various groups have shown that, at least under pathological conditions, neurogenesis can occur in brain areas other than SVZ and SGZ.

Neurogenic areas are defined by the presence of neurogenic cells and their neurogenesis-permissive microenvironment, which is likely to include both histological peculiarities and specific local soluble signals. The neurogenic behavior of the progenitor cells throughout adult life appears determined by environmental signals in their niche. Still, populations of neural stem cells (NSCs) with neurogenic potential have been reported to exist in the normal adult cortex, amygdala, dorsal vagal complex of the adult brainstem, area CA1 of the hippocampus, substantia nigra, spinal cord, hypothalamus, striatum and white matter tracts (see figure). Even though the generation of neurons from NSCs in vivo in these regions remains controversial, the majority of the available data support the view that the potential of the NSCs is restricted by inhibitory cues, thus defining these brain areas as being primarily non-neurogenic in nature.

Methodological Considerations for Studying Adult Neurogenesis

2011-10-01T17:45:00.000-07:00

There are many methodological issues to consider when assessing adult neurogenesis in vivo.

One issue is the method of identifying proliferating NSCs and their daughter cells. The earliest studies used [³H]-thymidine, which gets incorporated into the DNA during the S-phase of the cell cycle, labels dividing cells and their progeny in vivo and can be detected by autoradiography.

The introduction of the synthetic thymidine analogue 5-bromo-2'deoxyuridine (BrdU) substitutes for thymidine in newly synthetized DNA of proliferating cells. BrdU incorporated into DNA can then be easily visualized by immunohistochemistry using specific anti-BrdU antibodies. With this method it is possible to perform a quantitative analysis of proliferation, phenotypic differentiation, and survival of newborn cells by varying the time interval between the pulsed administration of BrdU and the perfusion of animals. Using this approach, one can estimate the number of cells in S phase at a particular time if brains are examined at a relatively short time (e.g. 1 hour) after a BrdU pulse; proliferating cells can be tracked through several anatomical divisions, when examined several days or weeks after a BrdU pulse. Even though BrdU labeling is considered the gold standard for detection of adult neurogenesis, it has several pitfalls. To provide unambiguous data for neurogenesis, one must demonstrate BrdU-co-localisation with phenotypic markers in three orthogonal planes using confocal microscopy. Additionally, sources of false positive phenomena need to be excluded (mitosis-like apoptosis, DNA repair, fusion of neurons with proliferating glia, DNA endo-replication). Although DNA labeling by BrdU is currently the most commonly used method for studying adult neurogenesis, the potential toxic effect of this thymidine analogue should not be ignored, as it might be a confounding factor in some experiments.

This has led to the use of other markers of the cell cycle to analyse cell proliferation in situ, such as proliferating nuclear antigen (PCNA) and Ki-67. However, since the expression of these markers is turned off after the termination of the mitotic cell cycle, the phenotypic differentiation of the newborn daughter cells can not be tracked.

To overcome this drawback, retroviral transfection may be used for labeling mitotic cells by forced expression of green fluorescent protein (GFP) or other reporter genes, thus allowing a complete morphological or electrophysiological analysis of the daughter cells (see video, unpublished data).

A second methodological issue concerns criteria for unambiguous identification of a cellular phenotype. Presently, the neuronal nuclei antigen (NeuN) is the standard immnunocytochemical marker to identify mature neurons. Glial fibrillary acidic protein (GFAP) and S100β are used as markers for mature astrocytes. However, at present there are no undisputed marker proteins that can provide unambiguous prospective identification of intermediate precursor cells in the adult brain.