A phase 2, double-blind, placebo-controlled study of NSI-189 phosphate, a neurogenic compound, among outpatients with major depressive disorder.

NSI-189 is a novel neurogenic compound independent of monoamine reuptake pathways. This trial evaluated oral NSI-189 as monotherapy in major depressive disorder. To improve signal detection, the sequential-parallel comparison design (SPCD) was chosen. Two hundred and twenty subjects were randomized to NSI-189 40 mg daily, 80 mg daily, or placebo for 12 weeks. The primary outcome measure was the Montogmery Asberg Depression Rating Scale (MADRS). Secondary subject-rated measures included the Symptoms of Depression Questionnaire (SDQ), the Cognitive and Physical Functioning Scale (CPFQ), the patient-rated version of the Quick Inventory of Depressive Symptomatology Scale (QIDS-SR), and subtests from the CogScreen and Cogstate cognitive tests. MADRS score reduction versus placebo did not reach significance for either dose (40 mg pooled mean difference -1.8, p = 0.22, 80 mg pooled mean difference -1.4, p = 0.34, respectively). However, the 40 mg dose showed greater overall reduction in SDQ (pooled mean difference -8.2; Cohen's d for Stages 1 and 2 = -0.11 and -0.64, p = 0.04), and CPFQ scores (pooled mean difference -1.9; Cohen's d for Stages 1 and 2 = -0.28 and -0.47, p = 0.03) versus placebo, as well as QIDS-SR scores in Stage 2 of SPCD (-2.5; Cohen's d Stages 1 and 2 = -0.03 and -0.68, p = 0.04). The 40 mg dose also showed advantages on some objective cognitive measures of the CogScreen (absolute Cohen's d ranged between 0.12 and 1.12 in favor of NSI-189, p values between 0.002 and 0.048 for those with overall significance), but not the Cogstate test. Both doses were well tolerated. These findings replicate those of phase 1b study, and warrant further exploration of the antidepressant and pro-cognitive effects of NSI-189.

A Phase 1B, randomized, double blind, placebo controlled, multiple-dose escalation study of NSI-189 phosphate, a neurogenic compound, in depressed patients.

We wanted to examine tolerability and efficacy of NSI-189, a benzylpiperizine-aminiopyridine neurogenic compound for treating major depressive disorder (MDD). This was a Phase 1B, double blind, randomized, placebo controlled, multiple-dose study with three cohorts. The first cohort received 40 mg q.d. (n=6) or placebo (n=2), the second cohort 40 mg b.i.d. (n=6) or placebo (n=2), and the third cohort 40 mg t.i.d. (n=6) or placebo (n=2). Twenty-four patients with MDD were recruited, with the diagnosis and severity confirmed through remote interviews. Eligible patients received NSI-189 or placebo for 28 days in an inpatient setting with assessments for safety, pharmacokinetics (PK) and efficacy. Outpatient follow-up visits were conducted until day 84 (±3). NSI-189 was relatively well tolerated at all doses, with no serious adverse effects. NSI-189 area under the curve increased in a dose-related and nearly proportional manner across the three cohorts, with a half-life of 17.4-20.5 h. The exploratory efficacy measurements, including Symptoms Of Depression Questionnaire (SDQ), Montgomery-Asberg Depression Scale (MADRS), Clinical Global Impressions-Improvement (CGI-I), and The Massachusetts General Hospital (MGH) Cognitive and Physical Functioning Questionnaire (CPFQ) showed a promising reduction in depressive and cognitive symptoms across all measures for NSI-189, with significant improvement in the SDQ and CPFQ, and a medium to large effect size for all measures. These improvements persisted during the follow-up phase. In summary, NSI-189 shows potential as a treatment for MDD in an early phase study. The main limitation of this preliminary study was the small sample size of each cohort.

Enhancement of Mitochondrial Function by the Neurogenic Molecule NSI-189 Accompanies Reversal of Peripheral Neuropathy and Memory Impairment in a Rat Model of Type 2 Diabetes.

Aims: Mitochondrial dysfunction contributes to many forms of peripheral and central nervous system degeneration. Therapies that protect mitochondrial number and function have the potential to impact the progression of conditions such as diabetic neuropathy. We therefore assessed indices of mitochondrial function in dorsal root ganglia (DRG) and brain cortex of the Zucker diabetic fatty (ZDF) rat model of type 2 diabetes and tested the therapeutic impact of a neurogenic compound, NSI-189, on both mitochondrial function and indices of peripheral and central neurological dysfunction. Materials and Methods: ZDF rats were maintained for 16 weeks of untreated diabetes before the start of oral treatment with NSI-189 for an additional 16 weeks. Nerve conduction velocity, sensitivity to tactile and thermal stimuli, and behavioral assays of cognitive function were assessed monthly. AMP-activated protein kinase (AMPK) phosphorylation, mitochondrial protein levels, and respiratory complex activities were assessed in the DRG and brain cortex after 16 weeks of treatment with NSI-189. Results: Treatment with NSI-189 selectively elevated the expression of protein subunits of complexes III and V and activities of respiratory complexes I and IV in the brain cortex, and this was accompanied by amelioration of impaired memory function and plasticity. In the sensory ganglia of ZDF rats, loss of AMPK activity was ameliorated by NSI-189, and this was accompanied by reversal of multiple indices of peripheral neuropathy. Conclusions: Efficacy of NSI-189 against dysfunction of the CNS and PNS function in type 2 diabetic rats was accompanied by improvement of mitochondrial function. NSI-189 exhibited actions at different levels of mitochondrial regulation in central and peripheral tissues.

Functions of basic fibroblast growth factor and neurotrophins in the differentiation of hippocampal neurons.

Restrictions in neuronal fate occur during the transition from a multipotential to a postmitotic cell. This and later steps in neuronal differentiation are determined by extracellular signals. We report that basic fibroblast growth factor is mitogenic for stem cells and is a differentiation factor for calbindin-expressing hippocampal neurons. The neurotrophin NT-3 is a differentiation factor for the same neurons but does not affect proliferation. NT-3 and brain-derived neurotrophic factor promote the maturation of neurons derived from stem cells that have been grown in vitro. These results define functions for basic fibroblast growth factor and neurotrophins in the differentiation processes that direct a multipotential stem cell to a specific neuronal fate.

Functional recovery in rats with ischemic paraplegia after spinal grafting of human spinal stem cells.

Transient spinal cord ischemia in humans can lead to the development of permanent paraplegia with prominent spasticity and rigidity. Histopathological analyses of spinal cords in animals with ischemic spastic paraplegia show a selective loss of small inhibitory interneurons in previously ischemic segments but with a continuing presence of ventral alpha-motoneurons and descending cortico-spinal and rubro-spinal projections. The aim of the present study was to examine the effect of human spinal stem cells (hSSCs) implanted spinally in rats with fully developed ischemic paraplegia on the recovery of motor function and corresponding changes in motor evoked potentials. In addition the optimal time frame for cell grafting after ischemia and the optimal dosing of grafted cells were also studied. Spinal cord ischemia was induced for 10 min using aortic occlusion and systemic hypotension. In the functional recovery study, hSSCs (10,000-30,000 cells/0.5 mul/injection) were grafted into spinal central gray matter of L2-L5 segments at 21 days after ischemia. Animals were immunosuppressed with Prograf (1 mg/kg or 3 mg/kg) for the duration of the study. After cell grafting the recovery of motor function was assessed periodically using the Basso, Beattie and Bresnahan (BBB) scoring system and correlated with the recovery of motor evoked potentials. At predetermined times after grafting (2-12 weeks), animals were perfusion-fixed and the survival, and maturation of implanted cells were analyzed using antibodies recognizing human-specific antigens: nuclear protein (hNUMA), neural cell adhesion molecule (hMOC), neuron-specific enolase (hNSE) and synapthophysin (hSYN) as well as the non-human specific antibodies TUJ1, GFAP, GABA, GAD65 and GLYT2. After cell grafting a time-dependent improvement in motor function and suppression of spasticity and rigidity was seen and this improvement correlated with the recovery of motor evoked potentials. Immunohistochemical analysis of grafted lumbar segments at 8 and 12 weeks after grafting revealed intense hNSE immunoreactivity, an extensive axo-dendritic outgrowth as well as rostrocaudal and dorsoventral migration of implanted hNUMA-positive cells. An intense hSYN immunoreactivity was identified within the grafts and in the vicinity of persisting alpha-motoneurons. On average, 64% of hSYN terminals were GAD65 immunoreactive which corresponded to GABA immunoreactivity identified in 40-45% of hNUMA-positive grafted cells. The most robust survival of grafted cells was seen when cells were grafted 21 days after ischemia. As defined by cell survival and laminar distribution, the optimal dose of injected cells was 10,000-30,000 cells per injection. These data indicate that spinal grafting of hSSCs can represent an effective therapy for patients with spinal ischemic paraplegia.

Age-dependent alterations in synaptic membrane systems for Ca2+ regulation.

The effects of aging on two neuronal plasma membrane Ca2+ regulating systems have been examined using synaptic membranes isolated from the brains of adult (5-7-month-old) and aged (23-25-month-old) Fisher 344 rats. The kinetic characteristics of the Na+-dependent Ca2+ transport system were found to be altered in the aged animals. The affinity of the transport carrier for Ca2+ was decreased in membranes from aged animals, with very little change in the maximal transport capacity of the system. The activity of the synaptic membrane Ca2+-activated, Mg2+-dependent ATPase was also altered in membranes from aged animals. In this system, however, the Vmax for Ca2+ activation of the enzymatic activity was lower in the aged animals, while there was no change in the K0.5 for Ca2+ activation. The magnitude of the alterations was small, but the differences were consistent. Even small changes in the effectiveness of the synaptic plasma membrane systems which participate in the maintenance of low intraterminal Ca2+ could progressively affect the integrity of synaptic transmission and lead eventually to neuronal cell death.

Studies on the ionic mechanism for the neuromodulatory actions of adenosine in the brain.

Although much evidence exists to support the modulation of neurotransmitter release by adenosine in both the central and peripheral nervous systems, the ionic mechanisms involved in this process are still not established. In our initial series of studies, the effects of adenosine analogs on depolarization-induced 45Ca influx into synaptosomes were examined, but no reduction in 45Ca uptake was observed in the presence of these agents, regardless of whether synaptosomes were depolarized by KCl or veratridine. The possibility that synaptosomal adenosine receptors are coupled directly to K+ channels and that an increase in K+ conductance is the primary ionic event initiated by adenosine was examined in resealed synaptic plasma membranes. We followed the movement of K+ through the use of the labeled lipophilic anion [35S]thiocyanate [( 35S]SCN-), which distributes across membranes according to the existing membrane potential. Both 2-chloroadenosine and adenosine-5'-cyclopropyl carboxamide produced dose-dependent increases in SCN- uptake, indicating enhanced K+ fluxes across the synaptic membranes. These effects of adenosine receptor agonists on membrane K+ conductance were blocked by receptor antagonists such as isobutylmethylxanthine. In addition, the placement of a K+-selective microelectrode in the caudate nuclei of intact, anesthetized rats revealed a substantial increase in the extracellular K+ concentration when adenosine was released onto the cells from a pipet in the assembly containing the ion-selective microelectrode. The results of these studies at both the membrane level and in the intact brain suggest that the initial event in the neuromodulatory actions of adenosine is an increase in the membrane conductance for K+ rather than inhibition of the voltage sensitive Ca2+ channels.

Synthetic peptides homologous to human glycophorins of the Miltenberger complex of variants of MNSs blood group system specify the epitopes for Hil, SJL, Hop, and Mur antisera.

The antigenic epitopes of the MNSs blood groups are localized on alpha and delta glycophorins (glycophorins A and B) of the erythrocyte surface. Hil, SJL, Mur, and Hop antisera define the Miltenberger (Mi) complex of MiV, MiJ.L., MiIII, and MiVI variant serologic phenotypes of this blood group system. We report here the location of the epitopes for antibodies in these antisera. The antigens of these Mi classes are variant glycophorins that are hybrids of alpha and delta glycophorins in alpha-delta and delta-alpha-delta arrangements. The hybrid junctions give rise to novel polypeptide sequences not present in the parent glycophorins; in MiIII and MiVI this also includes an expressed sequence of the delta pseudoexon. These sequences are identical in the above Mi-glycophorins occurring in erythrocytes that share a common Mi determinant. Four peptides of 10 to 14 amino acids each were constructed to be homologous to the identical sequences; they were designated, "Hil", "SJL", "Mur", and "Hop" to reflect the common determinant. The peptides were tested for inhibition of reaction of appropriate cells with the relevant antisera. The Hil peptide, outlining the alpha-delta s junction region in MiIII, MiV, and MiVI glycophorins, inhibited the reaction of respective erythrocytes (red blood cells [RBCs]) with anti-Hil. The SJL peptide, which differs from the Hil peptide by a single Thr----Met substitution, was specific for inhibition of the reaction of MiJ.L. RBCs with anti-SJL (an example of anti-S specific for such RBCs). The Hop peptide, which corresponds to the delta-alpha junction in MiVI glycophorin, inhibited the hemagglutination of MiVIII RBCs by anti-Hop. MiVI and MiVIII glycophorins share an identical sequence at that site. The Mur peptide, corresponding to a portion of the expressed pseudoexon sequence in MiIII and MiVI glycophorins, was specific for inhibition of the reaction of MiIII and MiVI RBCs with anti-Mur. The peptides had no effect on the hemagglutination of control MNSs RBCs by their respective antisera nor of unrelated Mi classes RBCs by antisera that distinguish these classes. We conclude that the alpha-delta junction in MiIII, MiV, and MiVI glycophorins outlines the epitopes for anti-Hil, the alpha-delta junction in MiJ.L. outlines the epitope for anti-SJL, the delta-alpha junction in MiVI constitutes the epitope for anti-Hop, and the expressed delta pseudoexon sequence in MiIII and MiVI constitutes the epitope for anti-Mur.

Biochemistry and molecular biology of MNSs blood group antigens.

This chapter has reviewed the nature of antigens of the MNSs blood group system. The structures of the proteins and the molecular features and organization of glycophorin genes were described, emphasizing their domain arrangement and the extensive sequence homology that indicates that their common and variant alleles belong to a single gene family. Methods currently used to examine these antigens are immunoblotting and DNA typing. The majority of variant genes are hybrids of parent glycophorin genes in a variety of arrangements; they contain no other sequences but those of the parent genes. The structures of the hybrids are summarized in Figure 8. Several hybrids appear to have arisen by unequal homologous recombination but others appear to have occurred through gene conversion. In this system the molecular genetic basis for a single variant phenotype may differ, as documented by gene rearrangements that appear to have occurred, as separate events, at different sites in the same intron; this has resulted in protein structures (hence phenotypes) that are identical. For example, unequal homologous recombination occurring within intron 3 can have given rise to only a limited number of phenotypes, namely alpha M-delta S, alpha N-delta S, alpha M-delta S, alpha N-delta S and delta-alpha. In addition, different sites of an exon may have been involved in gene rearrangements through gene conversion leading to nearly identical protein structures, yet different serological phenotypes. Thus, gene conversion could be more significant for generation of antigenic diversification as the number of possible new alleles is quite large. The participation of the HGpE gene in these rearrangements would make this number even larger. New sites and the expressed pseudoexon have created the epitopes of the variant phenotypes, and sequences specific for several variant antisera have been identified. Thus, the molecular basis for several serological reactions involving this system is now better understood.

Amino acid sequence of an alpha-delta-glycophorin hybrid. A structure reciprocal to Sta delta-alpha-glycophorin hybrid.

In this report we examine the primary sequence of a variant glycophorin obtained from erythrocytes of an individual who exhibits an unusual MNSs blood group phenotype. We show that this protein is a hybrid molecule constructed from sequences of alpha- and delta-glycophorins (glycophorins A and B) in a alpha-delta arrangement. Serological typing revealed that the donor's phenotype was M+N+S+s+U+; yet his erythrocytes reacted with some but not all examples of anti-S antisera. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a variant glycophorin band, and immunoblotting and reaction with N-glycanase suggested that its amino terminus resembled that of M-alpha-glycophorin but that its carboxyl terminus did not. A preparation highly enriched in the variant was obtained and used to generate peptide fragments for sequencing. The sequence revealed that the variant was a hybrid molecule whose amino terminus corresponded to M-alpha-glycophorin and whose carboxyl terminus corresponded to S-delta-glycophorin. CNBr cleavage of the variant glycophorin yielded four peptides. The sequence of the amino-terminal CNBr peptide (residues 1-8) was identical to the amino-terminal octapeptide of M-alpha-glycophorin. The proceeding peptide (residues 9-61) contained a segment identical to residues 9-58 of alpha glycophorin, but its carboxyl-terminal sequence had the Gly-Glu-Met sequence from S-delta-glycophorin (residues 27-29). The other two peptides, insoluble in aqueous solutions, contained highly hydrophobic sequences, identical to residues 30-52 and 53-68 of delta-glycophorin. Sequences of overlapping peptides generated by trypsin and V8 protease confirmed the hybrid nature of the variant glycophorin: residues 1-58 were identical to residues 1-58 of M-alpha-glycophorin, and residues 59-100 were entirely identical to residues 27-68 of S-delta-glycophorin. The variant glycophorin is expected to have 4 additional residues at its carboxyl terminus that correspond to the carboxyl-terminal residues 69-72 of delta-glycophorin. The amino acid sequence arrangement of the variant alpha-delta-glycophorin is an exact reciprocal of that found in another hybrid glycophorin, Sta, that is a delta-alpha hybrid. We propose that the two hybrid glycophorins represent the two possible products resulting from a reciprocal recombination event.

Delta glycophorin (glycophorin B) gene deletion in two individuals homozygous for the S--s--U-- blood group phenotype.

Blood cells from two unrelated individuals whose erythrocytes exhibit respectively N S-s-U- and MN S-s-U- blood group phenotypes were examined by immunoblotting, periodic acid-Schiff (PAS) staining, and Southern blotting. Protein bands characteristic of delta glycophorin (glycophorin B) were absent from the immunoblots of whole erythrocyte lysates when probed with polyclonal glycophorin antisera and from isolated erythrocyte membranes stained with PAS reagents. Genomic DNA from the two individuals' leukocytes was digested with a panel of restriction enzymes and probed with alpha M glycophorin cDNA obtained from human K562 leukemic cell line. The EcoRI, PstI, and KpnI restriction site patterns were identical to those of S+s+U+ controls in fragment numbers and relative size but differed from controls in band intensities. Restriction mapping with HindIII, PvuII, SacI, MspI, and BamHI revealed that S-s-U- individuals lack fragments that are reproducibly observed in S+s+U+ controls, and most likely encode delta glycophorin. Using truncated 5' and 3' cDNA segments as probes and comparing, in control individuals, hybridization intensities of fragments with amino acid sequence homologies, we have inferred the assignment of restriction fragments to the alpha and delta glycophorin genes. Our results suggest that the absence of delta glycophorin in the two S-s-U- individuals is a result of deletion of the entire delta glycophorin gene. This is the first report of a glycophorin gene deletion.

Single factors direct the differentiation of stem cells from the fetal and adult central nervous system.

Identifying the signals that regulate stem cell differentiation is fundamental to understanding cellular diversity in the brain. In this paper we identify factors that act in an instructive fashion to direct the differentiation of multipotential stem cells derived from the embryonic central nervous system (CNS). CNS stem cell clones differentiate to multiple fates: neurons, astrocytes, and oligodendrocytes. The differentiation of cells in a clone is influenced by extracellular signals: Platelet-derived growth factor (PDGF-AA, -AB, and -BB) supports neuronal differentiation. In contrast, ciliary neurotrophic factor and thyroid hormone T3 act instructively on stem cells to generate clones of astrocytes and oligodendrocytes, respectively. Adult stem cells had remarkably similar responses to these growth factors. These results support a simple model in which transient exposure to extrinsic factors acting through known pathways initiates fate decisions by multipotential CNS stem cells.