Outcomes of patients who underwent treatment for anti-HLA donor-specific antibodies before receiving a haploidentical hematopoietic cell transplant.

Pediatric and adolescent and young adult (AYA) patients who receive many blood product transfusions, such as individuals with sickle cell disease (SCD), severe aplastic anemia (SAA) or indolent hematologic malignancies, are at high risk for developing donor-specific antibodies (DSA). DSAs with mean fluorescence intensity (MFI) greater than 5000 have been associated with significant graft failure, but lower MFI values between 2000 and 5000 may result in poor graft function after hematopoietic cell transplant (HCT). Desensitization strategies have been developed to reduce the DSA burden in HCT recipients before graft infusion, but the experience with these strategies in the pediatric and AYA populations is not well described in the literature. Here, we describe our experience with successful desensitization by using a combination of treatment strategies in five pediatric and AYA patients, including a novel use of daratumumab in a young adult patient who had refractory DSAs and had suffered serious side effects from conventional desensitization strategies. The presence of elevated DSAs in pediatric and AYA recipients of a human leukocyte antigen (HLA)-mismatched haploidentical HCT can be overcome by a multipronged treatment strategy.

Type I interferon protects against pneumococcal invasive disease by inhibiting bacterial transmigration across the lung.

Streptococcus pneumoniae infection is a leading cause of bacterial pneumonia, sepsis and meningitis and is associated with high morbidity and mortality. Type I interferon (IFN-I), whose contribution to antiviral and intracellular bacterial immunity is well established, is also elicited during pneumococcal infection, yet its functional significance is not well defined. Here, we show that IFN-I plays an important role in the host defense against pneumococci by counteracting the transmigration of bacteria from the lung to the blood. Mice that lack the type I interferon receptor (Ifnar1 (-/-)) or mice that were treated with a neutralizing antibody against the type I interferon receptor, exhibited enhanced development of bacteremia following intranasal pneumococcal infection, while maintaining comparable bacterial numbers in the lung. In turn, treatment of mice with IFNß or IFN-I-inducing synthetic double stranded RNA (poly(I:C)), dramatically reduced the development of bacteremia following intranasal infection with S. pneumoniae. IFNß treatment led to upregulation of tight junction proteins and downregulation of the pneumococcal uptake receptor, platelet activating factor receptor (PAF receptor). In accordance with these findings, IFN-I reduced pneumococcal cell invasion and transmigration across epithelial and endothelial layers, and Ifnar1 (-/-) mice showed overall enhanced lung permeability. As such, our data identify IFN-I as an important component of the host immune defense that regulates two possible mechanisms involved in pneumococcal invasion, i.e. PAF receptor-mediated transcytosis and tight junction-dependent pericellular migration, ultimately limiting progression from a site-restricted lung infection to invasive, lethal disease.

NIK prevents the development of hypereosinophilic syndrome-like disease in mice independent of IKKα activation.

Immune cell-mediated tissue injury is a common feature of different inflammatory diseases, yet the pathogenetic mechanisms and cell types involved vary significantly. Hypereosinophilic syndrome (HES) represents a group of inflammatory diseases that is characterized by increased numbers of pathogenic eosinophilic granulocytes in the peripheral blood and diverse organs. On the basis of clinical and laboratory findings, various forms of HES have been defined, yet the molecular mechanism and potential signaling pathways that drive eosinophil expansion remain largely unknown. In this study, we show that mice deficient of the serine/threonine-specific protein kinase NF-κB-inducing kinase (NIK) develop a HES-like disease, reflected by progressive blood and tissue eosinophilia, tissue injury, and premature death at around 25-30 wk of age. Similar to the lymphocytic form of HES, CD4(+) T cells from NIK-deficient mice express increased levels of Th2-associated cytokines, and eosinophilia and survival of NIK-deficient mice could be prevented completely by genetic ablation of CD4(+) T cells. Experiments based on bone marrow chimeric mice, however, demonstrated that inflammation in NIK-deficient mice depended on radiation-resistant tissues, implicating that NIK-deficient immune cells mediate inflammation in a nonautonomous manner. Surprisingly, disease development was independent of NIK's known function as an IκB kinase α (IKKα) kinase, because mice carrying a mutation in the activation loop of IKKα, which is phosphorylated by NIK, did not develop inflammatory disease. Our data show that NIK activity in nonhematopoietic cells controls Th2 cell development and prevents eosinophil-driven inflammatory disease, most likely using a signaling pathway that operates independent of the known NIK substrate IKKα.

Residue Val237 is critical for the enantioselectivity of Penicillium expansum lipase.

The shape of the hydrophobic tunnel leading to the active site of Penicillium expansum lipase (PEL) was redesigned by single-point mutations, in order to better understand enzyme enantioselectivity towards naproxen. A variant with a valine-to-glycine substitution at residue 237 exhibited almost no enantioselectivity (E = 1.1) compared with that (E = 104) of wild-type PEL. The function of the residue, Val237, in the hydrophobic tunnel was further analyzed by site-directed mutagenesis. For each of these variants a significant decrease of enantioselectivity (E < 7) was observed compared with that of wild-type enzyme. Further docking result showed that Val237 plays the most important role in stabilizing the correct orientation of (R)-naproxen. Overall, these results indicate that the residue Val237 is the key amino acid residue maintaining the enantioselectivity of the lipase.

Donor-derived anti-HLA antibodies in a haploidentical hematopoietic cell transplant recipient shortly after transplant.

A pediatric patient with acute myeloid leukemia was referred to our institution for investigational therapy after disease relapse following a mismatched unrelated donor hematopoietic cell transplant (HCT). Prior to second HCT, the patient's serum was negative for antibodies to class I and class II HLA. Eight days after receiving a maternal donor haploidentical transplant, the patient became platelet refractory and highly sensitized to multiple class I HLA. Serum from the patient's mother was positive for the strongest antibodies present in the patient, suggesting the antibodies were donor-derived. Patient sera showed magnified and expanded sensitization over time in the context of 100% donor chimerism and despite undetectable circulating B cells. Escalating sensitization suggests active transfer of rituximab-resistant antibody-producing passenger lymphocytes from a haploidentical donor to a transplant recipient at the time of progenitor cell infusion. Evaluation of donor sensitization status may be a consideration prior to HLA mismatched HCT.

Substitution of Val72 residue alters the enantioselectivity and activity of Penicillium expansum lipase.

Error-prone PCR was used to create more active or enantioselective variants of Penicillium expansum lipase (PEL). A variant with a valine to glycine substitution at residue 72 in the lid structure exhibited higher activity and enantioselectivity than those of wild-type PEL. Site-directed saturation mutagenesis was used to explore the sequence-function relationship and the substitution of Val72 of P. expansum lipase changed both catalytic activity and enantioselectivity greatly. The variant V72A, displayed a highest enantioselectivity enhanced to about twofold for the resolution of (R, S)-naproxen (E value increased from 104 to 200.7 for wild-type PEL and V72A variant, respectively). In comparison to PEL, the variant V72A showed a remarkable increase in specific activity towards p-nitrophenyl palmitate (11- and 4-fold increase at 25 and 35 °C, respectively) whereas it had a decreased thermostability. The results suggest that the enantioselective variant V72A could be used for the production of pharmaceutical drugs such as enantiomerically pure (S)-naproxen and the residue Val 72 of P. expansum lipase plays a significant role in the enantioselectivity and activity of this enantioselective lipase.

The E3 ubiquitin ligase mind bomb-2 (MIB2) protein controls B-cell CLL/lymphoma 10 (BCL10)-dependent NF-κB activation.

B-cell CLL/lymphoma 10 (BCL10) is crucial for the activation of NF-κB in numerous immune receptor signaling pathways, including the T-cell receptor (TCR) and B-cell receptor signaling pathways. However, the molecular mechanisms that lead to signal transduction from BCL10 to downstream NF-κB effector kinases, such as TAK1 and components of the IKK complex, are not entirely understood. Here we used a proteomic approach and identified the E3 ligase MIB2 as a novel component of the activated BCL10 complex. In vitro translation and pulldown assays suggest direct interaction between BCL10 and MIB2. Overexpression experiments show that MIB2 controls BCL10-mediated activation of NF-κB by promoting autoubiquitination and ubiquitination of IKKγ/NEMO, as well as recruitment and activation of TAK1. Knockdown of MIB2 inhibited BCL10-dependent NF-κB activation. Together, our results identify MIB2 as a novel component of the activated BCL10 signaling complex and a missing link in the BCL10-dependent NF-κB signaling pathway.

Increased EID1 nuclear translocation impairs synaptic plasticity and memory function associated with pathogenesis of Alzheimer's disease.

Though loss of function in CBP/p300, a family of CREB-binding proteins, has been causally associated with a variety of human neurological disorders, such as Rubinstein-Taybi syndrome, Huntington's disease and drug addiction, the role of EP300 interacting inhibitor of differentiation 1 (EID1), a CBP/p300 inhibitory protein, in modulating neurological functions remains completely unknown. Through the examination of EID1 expression and cellular distribution, we discovered that there is a significant increase of EID1 nuclear translocation in the cortical neurons of Alzheimer's disease (AD) patient brains compared to that of control brains. To study the potential effects of EID1 on neurological functions associated with learning and memory, we generated a transgenic mouse model with a neuron-specific expression of human EID1 gene in the brain. Overexpression of EID1 led to an increase in its nuclear localization in neurons mimicking that seen in human AD brains. The transgenic mice had a disrupted neurofilament organization and increase of astrogliosis in the cortex and hippocampus. Furthermore, we demonstrated that overexpression of EID1 reduced hippocampal long-term potentiation and impaired spatial learning and memory function in the transgenic mice. Our results indicated that the negative effects of extra nuclear EID1 in transgenic mouse brains are likely due to its inhibitory function on CBP/p300 mediated histone and p53 acetylation, thus affecting the expression of downstream genes involved in the maintenance of neuronal structure and function. Together, our data raise the possibility that alteration of EID1 expression, particularly the increase of EID1 nuclear localization that inhibits CBP/p300 activity in neuronal cells, may play an important role in AD pathogenesis.

Bromodomain-Selective BET Inhibitors Are Potent Antitumor Agents against MYC-Driven Pediatric Cancer.

Inhibition of members of the bromodomain and extraterminal (BET) family of proteins has proven a valid strategy for cancer chemotherapy. All BET identified to date contain two bromodomains (BD; BD1 and BD2) that are necessary for recognition of acetylated lysine residues in the N-terminal regions of histones. Chemical matter that targets BET (BETi) also interact via these domains. Molecular and cellular data indicate that BD1 and BD2 have different biological roles depending upon their cellular context, with BD2 particularly associated with cancer. We have therefore pursued the development of BD2-selective molecules both as chemical probes and as potential leads for drug development. Here we report the structure-based generation of a novel series of tetrahydroquinoline analogs that exhibit >50-fold selectivity for BD2 versus BD1. This selective targeting resulted in engagement with BD-containing proteins in cells, resulting in modulation of MYC proteins and downstream targets. These compounds were potent cytotoxins toward numerous pediatric cancer cell lines and were minimally toxic to nontumorigenic cells. In addition, unlike the pan BETi (+)-JQ1, these BD2-selective inhibitors demonstrated no rebound expression effects. Finally, we report a pharmacokinetic-optimized, metabolically stable derivative that induced growth delay in a neuroblastoma xenograft model with minimal toxicity. We conclude that BD2-selective agents are valid candidates for antitumor drug design for pediatric malignancies driven by the MYC oncogene. SIGNIFICANCE: This study presents bromodomain-selective BET inhibitors that act as antitumor agents and demonstrates that these molecules have in vivo activity towards neuroblastoma, with essentially no toxicity.

Facile synthesis of 1,2-dione-containing abietane analogues for the generation of human carboxylesterase inhibitors.

Recently, a series of selective human carboxylesterase inhibitors have been identified based upon the tanshinones, with biologically active molecules containing a 1,2-dione group as part of a naphthoquinone core. Unfortunately, the synthesis of such compounds is complex. Here we describe a novel method for the generation of 1,2-dione containing diterpenoids using a unified approach, by which boronic acids are joined to vinyl bromo-cyclohexene derivatives via Suzuki coupling, followed by electrocyclization and oxidation to the o-phenanthroquinones. This has allowed the construction of a panel of miltirone analogues containing an array of substituents (methyl, isopropyl, fluorine, methoxy) which have been used to develop preliminary SAR with the two human carboxylesterase isoforms. As a consequence, we have synthesized highly potent inhibitors of these enzymes (Ki < 15 nM), that maintain the core tanshinone scaffold. Hence, we have developed a facile and reproducible method for the synthesis of abietane analogues that have resulted in a panel of miltirone derivatives that will be useful tool compounds to assess carboxylesterase biology.

Targeting ALK in pediatric RMS does not induce antitumor activity in vivo.

PURPOSE: The anaplastic lymphoma kinase (ALK) has been demonstrated to be a valid clinical target in diseases such as anaplastic large cell lymphoma and non-small cell lung cancer. Recent studies have indicated that ALK is overexpressed in pediatric rhabdomyosarcoma (RMS) and hence we hypothesized that this kinase may be a suitable candidate for therapeutic intervention in this tumor. METHODS: We evaluated the expression of ALK in a panel of pediatric RMS cell lines and patient-derived xenografts (PDX), and sensitivity to ALK inhibitors was assessed both in vitro and in vivo. RESULTS: Essentially, all RMS lines were sensitive to crizotinib, NVP-TAE684 or LDK-378 in vitro, and molecular analyses demonstrated inhibition of RMS cell proliferation following siRNA-mediated reduction of ALK expression. However, in vivo PDX studies using ALK kinase inhibitors demonstrated no antitumor activity when used as single agents or when combined with standard of care therapy (vincristine, actinomycin D and cyclophosphamide). More alarmingly, however, crizotinib actually accelerated the growth of these tumors in vivo. CONCLUSIONS: While ALK appears to be a relevant target in RMS in vitro, targeting this kinase in vivo yields no therapeutic efficacy, warranting extreme caution when considering the use of these agents in pediatric RMS patients.

Temporal response of neural progenitor cells to disease onset and progression in amyotrophic lateral sclerosis-like transgenic mice.

Regenerative medicine through neural stem cells (NSCs) or neural progenitor cells (NPCs) has been proposed as an alterative avenue for restoring neurological dysfunction in amyotrophic lateral sclerosis (ALS). It is critical to understand the organization and distribution of endogenous adult NPCs in response to motor neuron degeneration before regenerative medicine can be applied for ALS therapy. For this reason, we analyzed the temporal response of NPCs to motor neuron degeneration in the spinal cord and brain using nestin enhancer-driven LacZ reporter transgenic mice (pNes-Tg mice, control) and bi-transgenic mice containing both the nestin enhancer-driven LacZ reporter gene and mutant G93A-SOD1 gene (Bi-Tg mice). We observed an increase of NPCs in the dorsal horns of the spinal cord at the disease onset and progression stages in the Bi-Tg mice compared with that of age-matched pNes-Tg control mice. In contrast, an increase of NPCs in the ventral horns was detected at the disease progression stage. On the other hand, an increase of NPCs in the motor cortex at the disease-onset stage, but not at the disease progression stage, was detected. Furthermore, a decrease of NPCs in the lateral ventricle at the disease progression stage was observed, whereas no difference in the number of NPCs in the hippocampus was detected at the disease onset and progression stages. Some of the NPCs differentiate into neuron-like cells in response to motor neuron degeneration. The organization and distribution of endogenous adult NPCs in the ALS-like transgenic mice at the disease onset and progression stages provide fundamental bases for consideration of regenerative therapy of ALS by increasing de novo neurogenesis.

Selective Inhibitors of Human Liver Carboxylesterase Based on a ß-Lapachone Scaffold: Novel Reagents for Reaction Profiling.

Carboxylesterases (CEs) are ubiquitous enzymes that are responsible for the metabolism of xenobiotics, including drugs such as irinotecan and oseltamivir. Inhibition of CEs significantly modulates the efficacy of such agents. We report here that ß-lapachone is a potent, reversible CE inhibitor with Ki values in the nanomolar range. A series of amino and phenoxy analogues have been synthesized, and although the former are very poor inhibitors, the latter compounds are highly effective in modulating CE activity. Our data demonstrate that tautomerism of the amino derivatives to the imino forms likely accounts for their loss in biological activity. A series of N-methylated amino derivatives, which are unable to undergo such tautomerism, were equal in potency to the phenoxy analogues and demonstrated selectivity for the liver enzyme hCE1. These specific inhibitors, which are active in cell culture models, will be exceptionally useful reagents for reaction profiling of esterified drugs in complex biological samples.

Production of sorption functional media (SFM) from clinoptilolite tailings and its performance investigation in a biological aerated filter (BAF) reactor.

The few reuse and large stockpile of zeolite tailings led to a series of social and environmental problems. This study investigated the possibility of using the zeolite tailings as one of principal raw materials to prepare sorption functional media (SFM) by a high temperature sintering process. The SFM was used to serve as a biomedium in a biological aerated filter (BAF) reactor for domestic wastewater treatment, and its purification performance was examined. The physical, chemical and sorption properties of SFM were also determined. The microstructure of the SFM was analyzed by scanning electron microscopy (SEM). Results revealed that: (1) zeolite tailings could be used to produce the SFM under the optimal sintering parameters; (2) the sorption and desorption isotherm of ammonia nitrogen on SFM could be well described by the Langmuir formula; (3) in terms of removing organic matter, ammonia nitrogen, turbidity and colourity, the performance of the biofilter with SFM was superior to that with haydite; and (4) SFM BAF has a stronger adaptability to low temperature (6-11°C) for NH(3)-N removal compared to haydite BAF. Therefore, the SFM produced from the zeolite tailings was suitable to serve as the biomedium in the domestic wastewater treatment.

Neurogenic responses to amyloid-beta plaques in the brain of Alzheimer's disease-like transgenic (pPDGF-APPSw,Ind) mice.

Formation and accumulation of amyloid-beta (A beta) plaques are associated with declined memory and other neurocognitive function in Alzheimer's disease (AD) patients. However, the effects of A beta plaques on neural progenitor cells (NPCs) and neurogenesis from NPCs remain largely unknown. The existing data on neurogenesis in AD patients and AD-like animal models remain controversial. For this reason, we utilized the nestin second-intron enhancer controlled LacZ (pNes-LacZ) reporter transgenic mice (pNes-Tg) and Bi-transgenic mice (Bi-Tg) containing both pPDGF-APPSw,Ind and pNes-LacZ transgenes to investigate the effects of A beta plaques on neurogenesis in the hippocampus and other brain regions of the AD-like mice. We chose transgenic mice at 2, 8 and 12 months of age, corresponding to the stages of A beta plaque free, plaque onset and plaque progression to analyze the effects of A beta plaques on the distribution and de novo neurogenesis of (from) NPCs. We demonstrated a slight increase in the number of NPCs in the hippocampal regions at the A beta plaque free stage, while a significant decrease in the number of NPCs at A beta plaque onset and progression stages. On the other hand, we showed that A beta plaques increase neurogenesis, but not gliogenesis from post-mitotic NPCs in the hippocampus of Bi-Tg mice compared with age-matched control pNes-Tg mice. The neurogenic responses of NPCs to A beta plaques suggest that experimental approaches to promote de novo neurogenesis may potentially improve neurocognitive function and provide an effective therapy for AD.

Manganese superoxide dismutase protects mouse cortical neurons from chronic intermittent hypoxia-mediated oxidative damage.

Obstructive sleep apnea (OSA) syndrome has been recognized as a highly prevalent public health problem and is associated with major neurobehavioral morbidity. Chronic intermittent hypoxia (CIH), a major pathological component of OSA, increases oxidative damage to the brain cortex and decreases neurocognitive function in rodent models resembling human OSA. We employed in vitro and in vivo approaches to identify the specific phases and subcellular compartments in which enhanced reactive oxygen species (ROS) are generated during CIH. In addition, we utilized the cell culture and animal models to analyze the consequences of enhanced production of ROS on cortical neuronal cell damage and neurocognitive dysfunction. In a primary cortical neuron culture system, we demonstrated that the transition phase from hypoxia to normoxia (NOX) during CIH generates more ROS than the transition phase from NOX to hypoxia or hypoxia alone, all of which generate more ROS than NOX. Using selective inhibitors of the major pathways underlying ROS generation in the cell membrane, cytosol, and mitochondria, we showed that the mitochondria are the predominant source of enhanced ROS generation during CIH in mouse cortical neuronal cells. Furthermore, in both cell culture and transgenic mice, we demonstrated that overexpression of MnSOD-decreased CIH-mediated cortical neuronal apoptosis, and reduced spatial learning deficits measured with the Morris water maze assay. Together, the data from the in vitro and in vivo experiments indicate that CIH-mediated mitochondrial oxidative stress may play a major role in the neuronal cell loss and neurocognitive dysfunction in OSA. Thus, therapeutic strategies aiming at reducing ROS generation from mitochondria may improve the neurobehavioral morbidity in OSA.

Motor neuron degeneration promotes neural progenitor cell proliferation, migration, and neurogenesis in the spinal cords of amyotrophic lateral sclerosis mice.

The organization, distribution, and function of neural progenitor cells (NPCs) in the adult spinal cord during motor neuron degeneration in amyotrophic lateral sclerosis (ALS) remain largely unknown. Using nestin promoter-controlled LacZ reporter transgenic mice and mutant G93A-SOD1 transgenic mice mimicking ALS, we showed that there was an increase of NPC proliferation, migration, and neurogenesis in the lumbar region of adult spinal cord in response to motor neuron degeneration. The proliferation of NPCs detected by bromodeoxyurindine incorporation and LacZ staining was restricted to the ependymal zone surrounding the central canal (EZ). Once the NPCs moved out from the EZ, they lost the proliferative capability but maintained migratory function vigorously. During ALS-like disease onset and progression, NPCs in the EZ migrated initially toward the dorsal horn direction and then to the ventral horn regions, where motor neurons have degenerated. More significantly, there was an increased de novo neurogenesis from NPCs during ALS-like disease onset and progression. The enhanced proliferation, migration, and neurogenesis of (from) NPCs in the adult spinal cord of ALS-like mice may play an important role in attempting to repair the degenerated motor neurons and restore the dysfunctional circuitry which resulted from the pathogenesis of mutant SOD1 in ALS.

Early response of endogenous adult neural progenitor cells to acute spinal cord injury in mice.

Adult neural progenitor cells (NPCs) are an attractive source for functional replacement in neurodegenerative diseases and traumatic injury to the central nervous system (CNS). It has been shown that transplantation of neural stem cells or NPCs into the lesioned region partially restores CNS function. However, the capacity of endogenous NPCs in replacement of neuronal cell loss and functional recovery of spinal cord injury (SCI) is apparently poor. Furthermore, the temporal and spatial response of endogenous adult NPCs to SCI remains largely undefined. To this end, we have analyzed the early organization, distribution, and potential function of NPCs in response to SCI, using nestin enhancer (promoter) controlled LacZ reporter transgenic mice. We showed that there was an increase of NPC proliferation, migration, and neurogenesis in adult spinal cord after traumatic compression SCI. The proliferation of NPCs detected by 5-bromodeoxyuridine incorporation and LacZ staining was restricted to the ependymal zone (EZ) of the central canal. During acute SCI, NPCs in the EZ of the central canal migrated vigorously toward the dorsal direction, where the compression lesion is generated. The optimal NPC migration occurred in the adjacent region close to the epicenter. More significantly, there was an increased de novo neurogenesis from NPCs 24 hours after SCI. The enhanced proliferation, migration, and neurogenesis of (from) endogenous NPCs in the adult spinal cord in response to SCI suggest a potential role for NPCs in attempting to restore SCI-mediated neuronal dysfunction.

Enhanced de novo neurogenesis and dopaminergic neurogenesis in the substantia nigra of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson's disease-like mice.

Research reports on de novo neurogenesis, particularly dopaminergic (DA) neurogenesis in the adult mammalian substantia nigra (SN), remain very controversial. For this reason, we used the nestin second intron enhancer-controlled LacZ reporter transgenic mouse model coupled with the 1-methyl-4-phyenyl-1,2,3,6-tetrahydropyridine (MPTP) lesion system to investigate whether there are neurogenesis and DA neurogenesis in the SN of the adult normal and Parkinson's disease (PD)-like mice. First, we demonstrated the presence of neural progenitor cells (NPCs), basal levels of neurogenesis, and DA neurogenesis in the normal adult mouse SN. Second, we showed that there is not only a significant increase in the number of NPCs but also a dramatic increase of neurogenesis from the NPCs in the SN and the midline region adjacent to the SN of the PD-like mice compared with that of normal controls. More importantly, we also demonstrated that there is an increase of DA neurogenesis in the SN of the MPTP-lesioned mice. Third, we showed that the increased DA neurogenesis in the MPTP-lesioned mice was derived from the NPCs and 5-bromodeoxyuridine-positive cells, suggesting that multiple stem cell lineages may contribute to the enhanced neurogenesis in the adult SN. Taken together, these results establish that there are basal levels, albeit low, and increased levels of de novo neurogenesis and DA neurogenesis in the SN of the adult normal and PD-like mice, respectively. The increased NPCs in the MPTP-lesioned mice further suggest that experimental approaches to promote de novo neurogenesis may provide an effective therapy for PD by functional replacement of degenerated DA neurons.