Adult Primate Brain Research Articles

Neural cell isolation from adult macaques for high-throughput analyses and neurosphere cultures.

The low number of neural progenitor cells (NPCs) present in the adult and aged primate brains represents a challenge for generating high-yield and viable in vitro cultures of primary brain cells. Here we report a step-by-step approach for the fast and reproducible isolation of high-yield and viable primary brain cells, including mature neurons, immature cells and NPCs, from adult and aged macaques. We describe the anesthesia, transcardial perfusion and brain tissue preparation; the subsequent microdissection of the regions of interest and their enzymatic dissociation, leading to the separation of single cells. The cell isolation steps of our protocol can also be used for routine cell culturing, in particular for NPC expansion and differentiation, suitable for studies of hippocampal neurogenesis in the adult macaque brain. The purified primary brain cells are largely free from myelin debris and erythrocytes, paving the way for multiple downstream applications in vitro and in vivo. When combined with single-cell profiling techniques, this approach allows an unbiased and comprehensive mapping of cell states in the adult and aged macaque brain, which is needed to advance our understanding of human cognitive and neurological diseases. The neural cell isolation protocol requires 4 h and a team of four to six users with expertize in primary brain cell isolation to avoid tissue hypoxia during the time-sensitive steps of the procedure.

In vivo Direct Conversion of Astrocytes to Neurons Maybe a Potential Alternative Strategy for Neurodegenerative Diseases.

Partly because of extensions in lifespan, the incidence of neurodegenerative diseases is increasing, while there is no effective approach to slow or prevent neuronal degeneration. As we all know, neurons cannot self-regenerate and may not be replaced once being damaged or degenerated in human brain. Astrocytes are widely distributed in the central nervous system (CNS) and proliferate once CNS injury or neurodegeneration occur. Actually, direct reprogramming astrocytes into functional neurons has been attracting more and more attention in recent years. Human astrocytes can be successfully converted into neurons in vitro. Notably, in vivo direct reprogramming of astrocytes into functional neurons were achieved in the adult mouse and non-human primate brains. In this review, we briefly summarized in vivo direct reprogramming of astrocytes into functional neurons as regenerative strategies for CNS diseases, mainly focusing on neurodegenerative diseases such as Parkinson’s disease (PD), Alzheimer’s disease (AD), and Huntington’s disease (HD). We highlight and outline the advantages and challenges of direct neuronal reprogramming from astrocytes in vivo for future neuroregenerative medicine.

In vivo Neuroregeneration to Treat Ischemic Stroke Through NeuroD1 AAV-Based Gene Therapy in Adult Non-human Primates.

Stroke may cause severe death and disability but many clinical trials have failed in the past, partially because the lack of an effective method to regenerate new neurons after stroke. In this study, we report an in vivo neural regeneration approach through AAV NeuroD1-based gene therapy to repair damaged brains after ischemic stroke in adult non-human primates (NHPs). We demonstrate that ectopic expression of a neural transcription factor NeuroD1 in the reactive astrocytes after monkey cortical stroke can convert 90% of the infected astrocytes into neurons. Interestingly, astrocytes are not depleted in the NeuroD1-converted areas, consistent with the proliferative capability of astrocytes. Following ischemic stroke in monkey cortex, the NeuroD1-mediated astrocyte-to-neuron (AtN) conversion significantly increased local neuronal density, reduced microglia and macrophage, and surprisingly protected parvalbumin interneurons in the converted areas. Furthermore, the NeuroD1 gene therapy showed a broad time window in AtN conversion, from 10 to 30 days following ischemic stroke. The cortical astrocyte-converted neurons showed Tbr1+ cortical neuron identity, similar to our earlier findings in rodent animal models. Unexpectedly, NeuroD1 expression in converted neurons showed a significant decrease after 6 months of viral infection, indicating a downregulation of NeuroD1 after neuronal maturation in adult NHPs. These results suggest that in vivo cell conversion through NeuroD1-based gene therapy may be an effective approach to regenerate new neurons for tissue repair in adult primate brains.

Power spectrum analysis of EEG in a translational nonhuman primate model after chronic exposure to low levels of the common marine neurotoxin, domoic acid

Domoic acid (DA), the focus of this research, is a marine algal neurotoxin and epileptogen produced by species in the genus Pseudo-nitzschia. DA is found in finfish and shellfish across the globe. The current regulatory limit for DA consumption (20 ppm in shellfish) was set to protect humans from acute toxic effects, but there is a growing body of evidence suggesting that regular consumption of DA contaminated seafood at or below the regulatory limit may lead to subtle neurological effects in adults. The present research uses a translational nonhuman primate model to assess neurophysiological changes after chronic exposure to DA near the regulatory limit. Sedated electroencephalography (EEG) was used in 20 healthy adult female Macaca fascicularis, orally administered 0.075 and 0.15 mg DA/kg/day for at least 10 months. Paired video and EEG recordings were cleaned and a Fast Fourier Transformation was applied to EEG recordings to assess power differences in frequency bands from 1−20 Hz. When DA exposed animals were compared to controls, power was significantly decreased in the delta band (1−4 Hz, p < 0.005) and significantly increased in the alpha band (5−8 Hz, p < 0.005), theta band (9−12 Hz, p < 0.01), and beta band (13−20 Hz, p < 0.05). The power differences were not dose dependent or related to the duration of DA exposure, or subtle clinical symptoms of DA exposure (intentional tremors). Alterations of power in these bands have been associated with a host of clinical symptoms, such as deficits in memory and neurodegenerative diseases, and ultimately provide new insight into the subclinical toxicity of chronic, low-dose DA exposure on the adult primate brain.

Reduced Midbrain Dopamine Neuron Number in the Adult Non-human Primate Brain after Fetal Radiation Exposure

Early gestation is a neurodevelopmental period that is especially vulnerable to environmental insult and one in which neurogenesis features prominently. Prenatal perturbation during early gestation has been linked to neuropsychiatric illnesses such as autism and schizophrenia, and severe environmental insult during this period can result in profound mental impairment. Midbrain dopamine neurons are generated during early gestation and play a key role in the motor, cognitive and reward circuitries implicated in neuropsychiatric disease and addiction. This study examined the impact of curtailing neurogenesis in early gestation on neuron number in the midbrain dopamine group, i.e., the substantia nigra and contiguous ventral tegmental area. Rhesus macaque monkeys were exposed in utero on embryonic days 39–41 to x-irradiation (3–4 exposures of 50 cGy over 3–7 days totalling <200 cGy) and allowed to mature to full adulthood. Stereologic cell counts of tyrosine hydroxylase-positive neurons in the midbrain dopamine group were performed in adult monkeys, as were measurements of somal size. Mean total neuron number in the irradiated monkeys was significantly reduced on average by 33% compared to that of the control group. Somal size did not differ between the groups, suggesting that the integrity of survivor populations was not impacted. Reduced midbrain dopamine neuron number in fetally irradiated, adult monkeys indicates that radiation exposure during the critical period of neurogenesis results in an enduring reduction of this population and underscores the susceptibility of early neurodevelopmental processes to irreversible damage from environmental exposures.

Body map proto-organization in newborn macaques

Topographic sensory maps are a prominent feature of the adult primate brain. Here, we asked whether topographic representations of the body are present at birth. Using functional MRI (fMRI), we find that the newborn somatomotor system, spanning frontoparietal cortex and subcortex, comprises multiple topographic representations of the body. The organization of these large-scale body maps was indistinguishable from those in older monkeys. Finer-scale differentiation of individual fingers increased over the first 2 y, suggesting that topographic representations are refined during early development. Last, we found that somatomotor representations were unchanged in 2 visually impaired monkeys who relied on touch for interacting with their environment, demonstrating that massive shifts in early sensory experience in an otherwise anatomically intact brain are insufficient for driving cross-modal plasticity. We propose that a topographic scaffolding is present at birth that both directs and constrains experience-driven modifications throughout somatosensory and motor systems.

Premotor Cortical-Cerebellar Reorganization in a Macaque Model of Primary Motor Cortical Lesion and Recovery.

Neuromotor systems have the capacity for functional recovery following local damage. The literature suggests a possible role for the premotor cortex and cerebellum in motor recovery. However, the specific changes to interactions between these areas following damage remain unclear. Here, we demonstrate potential rewiring of connections from the ipsilesional ventral premotor cortex (ip-PMv) to cerebellar structures in a nonhuman primate model of primary motor cortex (M1) lesion and motor recovery. Cerebellar connections arising from the ip-PMv were investigated by comparing biotinylated dextran amine (BDA) between two groups of male Macaca mulatta: M1-lesion/motor recovery group and intact group. There were more BDA-labeled boutons and axons in all ipsilesional deep cerebellar nuclei (fastigial, interposed, and dentate) in the M1-lesion/recovery group than in the intact group. The difference was evident in the ipsilesional fastigial nucleus (ip-FN), and particularly observed in its middle, a putative somatosensory region of the ip-FN, which was characterized by absent or little expression of aldolase C. Some of the altered projections from the ip-PMv to ip-FN neurons were confirmed as functional because the synaptic markers, synaptophysin and vesicular glutamate transporter 1, were colocalized with BDA-labeled boutons. These results suggest that the adult primate brain after motor lesions can reorganize large-scale networks to enable motor recovery by enhancing sensorimotor coupling and motor commands via rewired fronto-cerebellar connections.SIGNIFICANCE STATEMENT Damaging the motor cortex causes motor deficits, which can be recovered over time. Such motor recovery may result from functional compensation in remaining neuromotor areas, including the ventral premotor cortex. We investigated compensatory changes in neural axonal outputs from ventral premotor to deep cerebellar nuclei in a monkey model of primary motor cortical lesion and motor recovery. The results showed an increase in premotor projections and synaptic formations in deep cerebellar nuclei, especially the sensorimotor region of the fastigial nucleus. Our results provide the first evidence that large-scale reorganization of fronto-cerebellar circuits may underlie functional recovery after motor cortical lesions.

Chronic, low-level oral exposure to marine toxin, domoic acid, alters whole brain morphometry in nonhuman primates

Domoic acid (DA) is an excitatory neurotoxin produced by marine algae and responsible for Amnesiac Shellfish Poisoning in humans. Current regulatory limits (˜0.075-0.1 mg/kg/day) protect against acute toxicity, but recent studies suggest that the chronic consumption of DA below the regulatory limit may produce subtle neurotoxicity in adults, including decrements in memory. As DA-algal blooms are increasing in both severity and frequency, we sought to better understand the effects of chronic DA exposure on reproductive and neurobehavioral endpoints in a preclinical nonhuman primate model. To this end, we initiated a long-term study using adult, female Macaca fascicularis monkeys exposed to daily, oral doses of 0.075 or 0.15 mg/kg of DA for a range of 321–381, and 346–554 days, respectively. This time period included a pre-pregnancy, pregnancy, and postpartum period. Throughout these times, trained data collectors observed intentional tremors in some exposed animals during biweekly clinical examinations. The present study explores the basis of this neurobehavioral finding with in vivo imaging techniques, including diffusion tensor magnetic resonance imaging and spectroscopy. Diffusion tensor analyses revealed that, while DA exposed macaques did not significantly differ from controls, increases in DA-related tremors were negatively correlated with fractional anisotropy, a measure of structural integrity, in the internal capsule, fornix, pons, and corpus callosum. Brain concentrations of lactate, a neurochemical closely linked with astrocytes, were also weakly, but positively associated with tremors. These findings are the first documented results suggesting that chronic oral exposure to DA at concentrations near the current human regulatory limit are related to structural and chemical changes in the adult primate brain.

The Subcellular Distribution of Alpha-Tocopherol in the Adult Primate Brain and Its Relationship with Membrane Arachidonic Acid and Its Oxidation Products.

The relationship between α-tocopherol, a known antioxidant, and polyunsaturated fatty acid (PUFA) oxidation, has not been directly investigated in the primate brain. This study characterized the membrane distribution of α-tocopherol in brain regions and investigated the association between membrane α-tocopherol and PUFA content, as well as brain PUFA oxidation products. Nuclear, myelin, mitochondrial, and neuronal membranes were isolated using a density gradient from the prefrontal cortex (PFC), cerebellum (CER), striatum (ST), and hippocampus (HC) of adult rhesus monkeys (n = 9), fed a stock diet containing vitamin E (α-, γ-tocopherol intake: ~0.7 µmol/kg body weight/day, ~5 µmol/kg body weight/day, respectively). α-tocopherol, PUFAs, and PUFA oxidation products were measured using high performance liquid chromatography (HPLC), gas chromatography (GC) and liquid chromatography-gas chromatography/mass spectrometry (LC-GC/MS) respectively. α-Tocopherol (ng/mg protein) was highest in nuclear membranes (p < 0.05) for all regions except HC. In PFC and ST, arachidonic acid (AA, µg/mg protein) had a similar membrane distribution to α-tocopherol. Total α-tocopherol concentrations were inversely associated with AA oxidation products (isoprostanes) (p < 0.05), but not docosahexaenoic acid oxidation products (neuroprostanes). This study reports novel data on α-tocopherol accumulation in primate brain regions and membranes and provides evidence that α-tocopherol and AA are similarly distributed in PFC and ST membranes, which may reflect a protective effect of α-tocopherol against AA oxidation.

Spatiotemporal patterns of cortical fiber density in developing infants, and their relationship with cortical thickness.

The intrinsic relationship between the convoluted cortical folding and the underlying complex whiter matter fiber connections has received increasing attention in current neuroscience studies. Recently, the axonal pushing hypothesis of cortical folding has been proposed to explain the finding that the axonal fibers (derived from diffusion tensor images) connecting to gyri are significantly denser than those connecting to sulci in both adult human and non-human primate brains. However, it is still unclear about the spatiotemporal patterns of the fiber density on the cortical surface of the developing infant brains from birth to 2 years of age, which is the most dynamic phase of postnatal brain development. In this paper, for the first time, we systemically characterized the spatial distributions and longitudinal developmental trajectories of the cortical fiber density in the first 2 postnatal years, via joint analysis of longitudinal structural and diffusion tensor imaging from 33 healthy infants. We found that the cortical fiber density increases dramatically in the first year and then keeps relatively stable in the second year. Moreover, we revealed that the cortical fiber density on gyral regions was significantly higher at 0, 1, and 2 years of age than that on sulcal regions in the frontal, temporal, and parietal lobes. Meanwhile, the cortical fiber density was strongly positively correlated with cortical thickness at several three-hinge junction regions of gyri. These results significantly advanced our understanding of the intrinsic relationship between the cortical folding, cortical thickness and axonal wiring during early postnatal stages.

Human neural stem cells survive long term in the midbrain of dopamine-depleted monkeys after GDNF overexpression and project neurites toward an appropriate target.

Transplanted multipotent human fetal neural stem cells (hfNSCs) significantly improved the function of parkinsonian monkeys in a prior study primarily by neuroprotection, with only 3%-5% of cells expressing a dopamine (DA) phenotype. In this paper, we sought to determine whether further manipulation of the neural microenvironment by overexpression of a developmentally critical molecule, glial cell-derived neurotrophic factor (GDNF), in the host striatum could enhance DA differentiation of hfNSCs injected into the substantia nigra and elicit growth of their axons to the GDNF-expressing target. hfNSCs were transplanted into the midbrain of 10 green monkeys exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine. GDNF was delivered concomitantly to the striatum via an adeno-associated virus serotype 5 vector, and the fate of grafted cells was assessed after 11 months. Donor cells remained predominantly within the midbrain at the injection site and sprouted numerous neurofilament-immunoreactive fibers that appeared to course rostrally toward the striatum in parallel with tyrosine hydroxylase-immunoreactive fibers from the host substantia nigra but did not mature into DA neurons. This work suggests that hfNSCs can generate neurons that project long fibers in the adult primate brain. However, in the absence of region-specific signals and despite GDNF overexpression, hfNSCs did not differentiate into mature DA neurons in large numbers. It is encouraging, however, that the adult primate brain appeared to retain axonal guidance cues. We believe that transplantation of stem cells, specifically instructed ex vivo to yield DA neurons, could lead to reconstruction of some portion of the nigrostriatal pathway and prove beneficial for the parkinsonian condition.

Huntington's disease: fighting on many fronts

The discovery in 1993 of the genetic abnormality that causes Huntington's disease triggered an explosion in our understanding of this inherited neurodegenerative disorder (The Huntington's Disease Collaborative Research Group, 1993). Since the Huntington's disease mutation is fully penetrant, everyone with the same underlying CAG triplet repeat expansion in the huntingtin ( HTT ) gene will develop a devastating combination of motor, cognitive and psychiatric disturbances. The monogenic nature of Huntington's disease suggests that disease modifying therapies should be within reach. Currently, however, no such treatments exist (Mestre et al. , 2009). Nevertheless, much has been done to characterize the huntingtin (Htt) protein and the effects of its toxic twin, mutant huntingtin. We know that Htt is a large and ubiquitously expressed protein with a range of possible functions, and that Huntington's disease is predominantly caused by the damaging effects of mutant Htt on neurons. A range of animal models recapitulate the molecular, cellular and clinical phenotypes of Huntington's disease, enhancing our understanding and facilitating the search for treatments (Ross and Tabrizi, 2011). Numerous potential pathogenic mechanisms have been identified, leading to promising therapeutic approaches in transgenic Huntington's disease mice and other models. Although none has yet been found effective in human patients, there is optimism in the field that Huntington's disease is a disease for which the goal of effective therapy might be achieved (Munoz-Sanjuan and Bates, 2011). Two articles in this issue of Brain highlight the Huntington's disease research community's multi-faceted approach to developing new treatments for Huntington's disease. Grondin and colleagues report success in a 6-month study of HTT gene silencing using RNA interference in the adult non-human primate brain (Grondin et al. , 2012); while Labbadia et al. (2012) describe the means by which a molecular chaperone can suppress aggregation of disease-associated Htt complexes. Gene silencing is an …

Six-month partial suppression of Huntingtin is well tolerated in the adult rhesus striatum.

Huntington's disease is caused by expression of a mutant form of Huntingtin protein containing an expanded polyglutamine repeat. One possible treatment for Huntington's disease may be to reduce expression of mutant Huntingtin in the brain via RNA interference. Unless the therapeutic molecule is designed to be allele-specific, both wild-type and mutant protein will be suppressed by an RNA interference treatment. A key question is whether suppression of wild-type as well as mutant Huntingtin in targeted brain regions can be tolerated and result in a net benefit to patients with Huntington's disease. Whether Huntingtin performs essential functions in the adult brain is unclear. Here, we tested the hypothesis that the adult primate brain can tolerate moderately reduced levels of wild-type Huntingtin protein for an extended period of time. A serotype 2 adeno-associated viral vector encoding for a short hairpin RNA targeting rhesus huntingtin messenger RNA (active vector) was bilaterally injected into the striatum of four adult rhesus monkeys. Four additional animals received a comparable vector encoding a scrambled control short hairpin RNA (control vector). General health and motor behaviour were monitored for 6 months. Upon termination, brain tissues were sampled and assessed blindly for (i) huntingtin messenger RNA knockdown; (ii) Huntingtin protein expression; and (iii) neuropathological changes. Reduction in wild-type huntingtin messenger RNA levels averaging ∼30% was measured in the striatum of active vector recipients 6 months post-injection. A widespread reduction in Huntingtin protein levels was also observed by immunohistochemistry in these animals, with an average protein reduction of ∼45% relative to controls measured by western blot analysis in the putamen of active vector recipients. As with control vector recipients, no adverse effects were observed behaviourally, and no neurodegeneration was found on histological examination of active vector recipients. Our results suggest that long-term partial suppression of wild-type Huntingtin may be safe, and thus if a comparable level of suppression of mutant Huntingtin is beneficial, then partial suppression of both wild-type and mutant Huntingtin may result in a net benefit in patients with heterozygous Huntington's disease.

Brain ischemia, neurogenesis, and neurotrophic receptor expression in primates.

Generation of new neurons persists in the normal adult mammalian brain, with neural stem/progenitor cells residing in at least two brain regions: the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus (DG). Adult neurogenesis is well documented in the rodent, and has also been demonstrated in vivo in nonhuman primates and humans. Brain injuries such as ischemia affect neurogenesis in adult rodents as both global and focal ischemic insults enhance the proliferation of progenitor cells residing in SGZ or SVZ. We addressed the issue whether an injury triggered activation of endogenous neuronal precursors also takes place in the adult primate brain. We found that the ischemic insult increased the number of progenitor cells in monkey SGZ and SVZ, and caused gliogenesis in the ischemia-prone hippocampal CA1 sector. To better understand the mechanisms regulating precursor cell division and differentiation in the primate, we analyzed the expression at protein level of a panel of potential regulatory molecules, including neurotrophic factors and their receptors. We found that a fraction of mitotic progenitors were positive for the neurotrophin receptor TrkB, while immature neurons expressed the neurotrophin receptor TrkA. Astroglia, ependymal cells and blood vessels in SVZ were positive for distinctive sets of ligands/receptors, which we characterized. Thus, a network of neurotrophic signals operating in an autocrine or paracrine manner may regulate neurogenesis in adult primate SVZ. We also analyzed microglial and astroglial proliferation in postischemic hippocampal CA1 sector. We found that proliferating postischemic microglia in adult monkey CA1 sector express the neurotrophin receptor TrkA, while activated astrocytes were labeled for nerve growth factor (NGF), ligand for TrkA, and the tyrosine kinase TrkB, a receptor for brain derived neurotrophic factor (BDNF). These results implicate NGF and BDNF as regulators of postischemic glial proliferation in adult primate hippocampus.

Expression of fatty acid‐binding proteins in adult hippocampal neurogenic niche of postischemic monkeys

Intracellular fatty acid (FA) chaperones known as FA-binding proteins (FABPs) are a group of molecules known to participate in cellular metabolic processes such as lipid storage, membrane synthesis, and β-oxidation or to coordinate transcriptional programs. However, their role in adult neurogenesis still remains obscure. The FABPs expressed in the central nervous system (CNS) are heart-type (FABP3), epidermal-type (FABP5), and brain-type (FABP7). These three FABPs possess a differential affinity for polyunsaturated fatty acids (PUFAs). Recently, we reported that GPR40, a receptor for free FAs and particularly for PUFAs, is expressed in the CNS of adult monkeys and upregulated after transient global brain ischemia in the hippocampal subgranular zone (SGZ), a neurogenic niche in adulthood. The SGZ showed a peak proliferation of progenitor cells and maximal expression of GPR40 during the second week after ischemia. As both FABPs and GPR40 might be closely related to the adult neurogenesis, here, we studied the expression of FABP 3, 5, and 7 in the SGZ, comparing normal and postischemic adult monkeys. Immunoblotting revealed that FABP5 and FABP7, but not FABP3, were significantly increased on day 15 after ischemia when compared with the nonischemic control. Immunohistochemistry showed that FABP5 was almost undetectable in the control SGZ but was abundant on day 15 after ischemia. FABP 3, 5, and 7 were expressed in S-100β-positive astrocytes and nestin-positive neural progenitors. However, only FABP 5 and 7 were found in bromodeoxyuridine (BrdU)-positive newly generated cells. FABPs were most frequently coexpressed with the S-100β-positive astrocytes, whereas βIII-tubulin-or polysialylated neural cell-adhesion molecule (PSA-NCAM)-positive newborn neurons in the vicinity of the astrocytes expressed none of the three FABPs. These results support a role of astrocyte- and/or neural progenitor-derived FABPs as components of the molecular machine regulating the progenitor cell niche in the adult primate brain.

Robust In Vivo Transduction of Nervous System and Neural Stem Cells by Early Gestational Intra Amniotic Gene Transfer Using Lentiviral Vector

Presently, in vivo methods to efficiently and broadly transduce all major cell types throughout both the central (CNS) and peripheral adult nervous system (PNS) are lacking. In this study, we hypothesized that during early fetal development neural cell populations, including neural stem cells (NSCs), may be accessible for gene transfer via the open neural groove. To test this hypothesis, we injected lentiviral vectors encoding a green fluorescent protein (GFP) marker gene into the murine amniotic cavity at embryonic day 8. This method (i) efficiently and stably transduced the entire nervous system for at least 80% of the lifespan of the mice, (ii) transduced all major neural cell types, and (iii) transduced adult NSCs of the subventricular zone (SVZ) and subgranular zones (SGZs). This simple approach has broad applications for the study of gene function in nervous system development and adult NSCs and may have future clinical applications for treatment of genetic disorders of the nervous system.

Endogenous Neural Stem Cells in the Adult Brain

Despite progress in our understanding molecular mechanisms of neuronal cell death in many central nervous system (CNS) diseases, widely effective treatments remain elusive. Recent studies have shown that neural stem cells (NSCs) are present in the subventricular zone (SVZ) lining the lateral ventricles and the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG) in adult mouse, rat, nonhuman primate, and human brain. Newly generated cells in the SGZ can differentiate into mature, functional neurons and integrate into the DG as granule cells, which are involved in memory formation. In addition, many CNS diseases can stimulate the proliferation of neuronal stem/progenitor cells located in the SVZ and SGZ of the adult rodent brain, and the resulting newborn cells migrate into damaged brain regions, where they express mature neuronal markers. Therefore, it might be possible for damaged cells to be replaced from endogenous neural stem cell pools. However, the capacity of self-repair is obviously not enough. Proliferation, migration, and neuronal differentiation of endogenous NSCs could be manipulated by pharmaceutical tools to reach the adequate benefits for the treatment of CNS diseases.

Immunohistochemical Distribution of NGF, BDNF, NT-3, and NT-4 in Adult Rhesus Monkey Brains

Immunohistochemical distribution and cellular localization of neurotrophins was investigated in adult monkey brains using antisera against nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). Western blot analysis showed that each antibody specifically recognized appropriate bands of approximately 14.7 kDa, 14.2 kDa, 13.6 kDa, and 14.5 kDa, for NGF, BDNF, NT-3, and NT-4, respectively. These positions coincided with the molecular masses of the neurotrophins studied. Furthermore, sections exposed to primary antiserum preadsorbed with full-length NGF, BDNF, NT-3, and NT-4 exhibited no detectable immunoreactivity, demonstrating specificities of the antibodies against the tissues prepared from rhesus monkeys. The study provided a systematic report on the distribution of NGF, BDNF, NT-3, and NT-4 in the monkey brain. Varying intensity of immunostaining was observed in the somata and processes of a wide variety of neurons and glial cells in the cerebrum, cerebellum, hippocampus, and other regions of the brain. Neurons in some regions such as the cerebral cortex and the hippocampus, which stained for neurotrophins, also expressed neurotrophic factor mRNA. In some other brain regions, there was discrepancy of protein distribution and mRNA expression reported previously, indicating a retrograde or anterograde action mode of neurotrophins. Results of this study provide a morphological basis for the elucidation of the roles of NGF, BDNF, NT-3, and NT-4 in adult primate brains.

Expression of angiogenic and neurotrophic factors in the progenitor cell niche of adult monkey subventricular zone

The subventricular zone along the anterior horn (SVZa) of the cerebral lateral ventricle of adult mammals contains multipotent progenitor cells, which supposedly exist in an angiogenic niche. Numerous signals are known to modulate the precursor cell proliferation, migration or differentiation, in rodent models. In contrast, the data on signals regulating the primate SVZa precursors in vivo are scarce. We analyzed the expression at protein level of a panel of angiogenic and/or neurotrophic factors and their receptors in SVZa of adult macaque monkeys, under normal condition or after transient global ischemia which enhances endogenous progenitor cell proliferation. We found that fms-like tyrosine kinase 1 (Flt1), a receptor for vascular endothelial cell growth factor, was expressed by over 30% of the proliferating progenitors, and the number of Flt1-positive precursors was significantly increased by the ischemic insult. Smaller fractions of mitotic progenitors were positive for the neurotrophin receptor tropomyosin-related kinase (Trk) B or the hematopoietic receptor Kit, while immature neurons expressed Flt1 and the neurotrophin receptor TrkA. Further, SVZa astroglia, ependymal cells and blood vessels were positive for distinctive sets of ligands/receptors, which we characterized. The presented data provide a molecular phenotypic analysis of cell types comprising adult monkey SVZa, and suggest that a complex network of angiogenic/neurotrophic signals operating in an autocrine or paracrine manner may regulate SVZa neurogenesis in the adult primate brain.

Immunohistochemical analysis of subcortical white matter astroglia of infant and adult primate brains, with a note on resident neurons

An immunohistochemical analysis of brain subcortical white matter astroglia from human (infant, adult) and adult monkey ( Cebus apella, Macaca nemestrina) cases without any known neurological disease, is described. Expression of synaptic vesicle-associated proteins, excitatory amino acid transporters (EAAT1 and EAAT2) and GABA A Rα 2 receptor produced coarse punctate labeling in human adult white matter astrocytes. A finer, generalized, punctate labeling was observed in human infants and adult C. apella monkeys. Labeling of neuronal somata and processes with microtubule-associated proteins (MAP2 a–c) and neuron nuclear (NeuN) antibodies, was also observed in subcortical white matter of humans and monkeys. Results suggest competence of subcortical white matter astroglia of the primate brain to participate in various transmitter regulatory pathways. It is also proposed that, collectively with resident neurons, they may exert some role in affecting the transfer of information that takes place through the various associational and projecting fiber systems coursing through this brain compartment.