Stephen M. Massa

Small molecule BDNF mimetics activate TrkB signaling and prevent neuronal degeneration in rodents

Brain-derived neurotrophic factor (BDNF) activates the receptor tropomyosin-related kinase B (TrkB) with high potency and specificity, promoting neuronal survival, differentiation, and synaptic function. Correlations between altered BDNF expression and/or function and mechanism(s) underlying numerous neurodegenerative conditions, including Alzheimer disease and traumatic brain injury, suggest that TrkB agonists might have therapeutic potential. Using in silico screening with a BDNF loop-domain pharmacophore, followed by low-throughput in vitro screening in mouse fetal hippocampal neurons, we have efficiently identified small molecules with nanomolar neurotrophic activity specific to TrkB versus other Trk family members. Neurotrophic activity was dependent on TrkB and its downstream targets, although compound-induced signaling activation kinetics differed from those triggered by BDNF. A selected prototype compound demonstrated binding specificity to the extracellular domain of TrkB. In in vitro models of neurodegenerative disease, it prevented neuronal degeneration with efficacy equal to that of BDNF, and when administered in vivo, it caused hippocampal and striatal TrkB activation in mice and improved motor learning after traumatic brain injury in rats. These studies demonstrate the utility of loop modeling in drug discovery and reveal what we believe to be the first reported small molecules derived from a targeted BDNF domain that specifically activate TrkB.We propose that these compounds constitute a novel group of tools for the study of TrkB signaling and may provide leads for developing new therapeutic agents for neurodegenerative diseases.

Acute neurologic and psychiatric complications associated with cocaine abuse.

This report reviewed 996 emergency room visits and 279 hospital admissions of patients with complications of cocaine abuse seen at the San Francisco General Hospital between 1979 and 1986. In 143 cases, acute neurologic or psychiatric symptoms were the primary complaint, and case-notes provided sufficient detail for analysis. The major neurologic complications included one or more seizures (n = 29), focal neurologic symptoms or signs (12), headache (10), and transient loss of consciousness (six). Psychiatric disturbances included agitation, anxiety, or depression (33), psychosis and paranoia (24), and suicidal ideation (18). The most serious consequences were found in patients with prolonged seizures or strokes, those who jumped out of buildings, and those who attempted suicide by overdosing with other drugs. There was no correlation between the appearance of complications and the reported route of administration, the amount of cocaine used, or prior experience with cocaine. The number of patients who are seeking hospital attention for these or related complaints appears to be rising substantially. Cocaine abuse, regardless of the use pattern, is associated with a variety of potentially severe neurologic and psychiatric complications.

Heme-oxygenase-1 induction in glia throughout rat brain following experimental subarachnoid hemorrhage

The heme released following subarachnoid hemorrhage is metabolized by heme-oxygenase (HO) to biliverdin and carbon monoxide (CO) with the release of iron. The HO reaction is important since heme may contribute to vasospasm and increase oxidative stress in cells. HO is comprised of at least two isozymes, HO-2 and HO-1. HO-1, also known as heat shock protein HSP32, is inducible by many factors including heme and heat shock. HO-2 does not respond to these stresses. To begin to examine HO activity following subarachnoid hemorrhage (SAH), the expression of HO-1 and HO-2 was investigated after experimental SAH in adult rats. Immunocytochemistry for HO-1, HO-2 and HSP70 proteins was performed at 1, 2, 3 and 4 days after injections of lysed blood, whole blood, oxyhemoglobin and saline into the cisterna magna. A large increase in HO-1 immunoreactivity was seen in cells throughout brain following injections of lysed blood, whole blood, and oxyhemoglobin but not saline. Lysed blood, whole blood and oxyhemoglobin induced HO-1 in all of the cortex, hippocampus, striatum, thalamus, forebrain white matter and in cerebellar cortex. HO-1 immunoreactivity was greatest in those regions adjacent to the basal subarachnoid cisterns where blood and oxyhemoglobin concentrations were likely highest. Double immunofluorescence studies showed the HO-1 positive cells to be predominately microglia, though HO-1 was induced in some astrocytes. HO-1 expression resolved by 48 h. HO-2 immunoreactivity was abundant but did not change following injections of blood. A generalized induction of HSP70 heat shock protein was not observed following injections of lysed blood, whole blood, oxyhemoglobin, or saline. These results suggest that HO-1 is induced in microglia throughout rat brain as a general, parenchymal response to the presence of oxyhemoglobin in the subarachnoid space and not as a stress response. This microglial HO-1 response could be protective against the lipid peroxidation and vasospasm induced by hemoglobin, by increasing heme clearance and iron sequestration, and enhancing the production of the antioxidant bilirubin.

Heme oxygenase-1 (HO-1) protein induction in rat brain following focal ischemia

The induction of the heme oxygenase-1 (HO-1) protein, also called HSP32, was compared to HSP70 heat shock protein induction following focal ischemia. Adult Sprague-Dawley male rats (n = 14) were subjected to either 30 min or 2 h of focal cerebral ischemia using the suture, middle-cerebral-artery (MCA) occlusion model. Controls (n = 4) had sham surgery. Following 24 h of reperfusion, subjects were killed and their brains stained immunocytochemically for HO-1 and the HSP70 heat shock proteins. One day following 30 min of ischemia, HO-1 and HSP70 staining in striatum occurred mainly in endothelial cells in infarcts and in glial cells surrounding the areas of infarction. Following the 30 min ischemia HO-1 was not induced in cortex whereas HSP70 was induced in cortical neurons in the MCA distribution. One day following 2 h of MCA ischemia, both HO-1 and HSP70 were induced in neurons in cortex in the MCA distribution. HO-1, however, was induced in glial cells throughout ipsilateral cortex, inside as well as outside the MCA distribution. This suggests that translation and/or transcription of the HO-1 and HSP70 genes are blocked in neurons and glia destined to die within infarcts, whereas translation of these stress genes continues in the endothelial cells. The duration of ischemia required to induce HSP70 in cortical neurons appears to be less than that required to induce HO-1 in cortical glia. Prolonged spreading depression and/or diffuse hemispheric ischemia may induce HO-1 in glia throughout the ipsilateral cortex via immediate early gene activation of the AP-1 site in the HO-1 promoter. Since HO-1 degrades heme, a pro-oxidant, to antioxidant molecules, the induction of HO-1 may augment oxidative defense mechanisms compromised by cerebral ischemia.

Global ischemia induces apoptosis-associated genes in hippocampus

Using in situ hybridization, Northern blotting and RT-PCR we studied the post-ischemic expression of bcl-2, bcl-x, bax and ICE. One day following 5 min or 10 min of global ischemia bcl-2 and bcl-x mRNAs were induced in CA1 hippocampal pyramidal neurons while bax was unchanged. By 72 h after ischemia the expression of bcl-2, bcl-x and bax mRNAs decreased in CA1. The large isoform of bcl-x (bcl-xL), detected using RT-PCR, decreased in whole hippocampus by 24-72 h after ischemia relative to the putative short (bcl-xS) and transmembrane deleted (bcl-x delta TM) forms. Oligonucleotides to interleukin-1 beta convertase (ICE), which detected the expected 2-kb transcript and two lesser 1.5- and 3-kb hybridizing species, demonstrated slight mRNA induction in the CA1 region at 72 h following ischemia. DNA nick end-labeling at 3 days following ischemia showed DNA fragmentation in neurons limited to the CA1 region of hippocampus following 5 min ischemia, while DNA fragmentation was detected in CA1, CA3, dentate gyrus and cortical neurons following 10 min ischemia. The data support the view that hippocampal neurons might undergo an apoptosis-like death after global ischemia. Since global ischemia decreases total protein synthesis especially in the CA1 region, the increases in bcl-2 mRNA levels may not necessarily lead to increased Bcl-2 protein levels. This may explain why the CA1 neurons die despite the prominent induction of the protective bcl-2 gene. The observed decrease by 24 h in the bcl-xL/bcl-xS ratio which preceded DNA fragmentation may participate in the cell death produced by ischemia. However, because of the ischemia-induced decrease in total protein synthesis, the decreased bcl-xL/bcl-xS ratio does not necessarily lead to a changed ratio in the amount of the appropriate proteins. Since ICE-like mRNA was induced at 72 h when the CA1 neurons were dead, the significance of this ICE-like mRNA induction remains unclear.

Deficient LAR expression decreases basal forebrain cholinergic neuronal size and hippocampal cholinergic innervation

A role in neural development for protein tyrosine phosphatase (PTPase) receptors has been suggested by the finding of aberrant neurite outgrowth in Drosophila mutants lacking functional leukocyte common antigen‐related (LAR) PTPase receptors; however, PTPase functions in the mammalian nervous system remain to be established. In transgenic mice containing a gene trap in the LAR gene, only trace expression of full‐length LAR transcripts was found. In these mice, the size of basal forebrain cholinergic neurons was significantly reduced and cholinergic innervation of the dentate gyrus was markedly decreased. These findings constitute the first demonstration of an aberrant neuronal phenotype in a mammalian PTPase mutant and support the hypothesis that LAR‐type PTPase receptors function to establish and/or maintain neuronal networks. J. Neurosci. Res. 47:348–360, 1997.

Small-molecule modulation of neurotrophin receptors: a strategy for the treatment of neurological disease

Neurotrophins and their receptors modulate multiple signalling pathways to regulate neuronal survival and to maintain axonal and dendritic networks and synaptic plasticity. Neurotrophins have potential for the treatment of neurological diseases. However, their therapeutic application has been limited owing to their poor plasma stability, restricted nervous system penetration and, importantly, the pleiotropic actions that derive from their concomitant binding to multiple receptors. One strategy to overcome these limitations is to target individual neurotrophin receptors — such as tropomyosin receptor kinase A (TRKA), TRKB, TRKC, the p75 neurotrophin receptor or sortilin — with small-molecule ligands. Such small molecules might also modulate various aspects of these signalling pathways in ways that are distinct from the programmes triggered by native neurotrophins. By departing from conventional neurotrophin signalling, these ligands might provide novel therapeutic options for a broad range of neurological indications.

The p75 neurotrophin receptor promotes amyloid-beta(1-42)-induced neuritic dystrophy in vitro and in vivo.

Oligomeric forms of amyloid-β (Aβ) are thought to play a causal role in Alzheimers disease (AD), and the p75 neurotrophin receptor (p75NTR) has been implicated in Aβ-induced neurodegeneration. To further define the functions of p75NTR in AD, we examined the interaction of oligomeric Aβ(1-42) with p75NTR, and the effects of that interaction on neurite integrity in neuron cultures and in a chronic AD mouse model. Atomic force microscopy was used to ascertain the aggregated state of Aβ, and fluorescence resonance energy transfer analysis revealed that Aβ oligomers interact with the extracellular domain of p75NTR. In vitro studies of Aβ-induced death in neuron cultures isolated from wild-type and p75NTR−/− mice, in which the p75NTR extracellular domain is deleted, showed reduced sensitivity of mutant cells to Aβ-induced cell death. Interestingly, Aβ-induced neuritic dystrophy and activation of c-Jun, a known mediator of Aβ-induced deleterious signaling, were completely prevented in p75NTR−/− neuron cultures. Thy1-hAPPLond/Swe × p75NTR−/− mice exhibited significantly diminished hippocampal neuritic dystrophy and complete reversal of basal forebrain cholinergic neurite degeneration relative to those expressing wild-type p75NTR. Aβ levels were not affected, suggesting that removal of p75NTR extracellular domain reduced the ability of excess Aβ to promote neuritic degeneration. These findings indicate that although p75NTR likely does not mediate all Aβ effects, it does play a significant role in enabling Aβ-induced neurodegeneration in vitro and in vivo, establishing p75NTR as an important therapeutic target for AD.

A TrkB Small Molecule Partial Agonist Rescues TrkB Phosphorylation Deficits and Improves Respiratory Function in a Mouse Model of Rett Syndrome

Rett syndrome (RTT) results from loss-of-function mutations in the gene encoding the methyl-CpG-binding protein 2 (MeCP2) and is characterized by abnormal motor, respiratory and autonomic control, cognitive impairment, autistic-like behaviors and increased risk of seizures. RTT patients and Mecp2-null mice exhibit reduced expression of brain-derived neurotrophic factor (BDNF), which has been linked in mice to increased respiratory frequency, a hallmark of RTT. The present study was undertaken to test the hypotheses that BDNF deficits in Mecp2 mutants are associated with reduced activation of the BDNF receptor, TrkB, and that pharmacologic activation of TrkB would improve respiratory function. We characterized BDNF protein expression, TrkB activation and respiration in heterozygous female Mecp2 mutant mice (Het), a model that recapitulates the somatic mosaicism for mutant MECP2 found in typical RTT patients, and evaluated the ability of a small molecule TrkB agonist, LM22A-4, to ameliorate biochemical and functional abnormalities in these animals. We found that Het mice exhibit (1) reduced BDNF expression and TrkB activation in the medulla and pons and (2) breathing dysfunction, characterized by increased frequency due to periods of tachypnea, and increased apneas, as in RTT patients. Treatment of Het mice with LM22A-4 for 4 weeks rescued wild-type levels of TrkB phosphorylation in the medulla and pons and restored wild-type breathing frequency. These data provide new insight into the role of BDNF signaling deficits in the pathophysiology of RTT and highlight TrkB as a possible therapeutic target in this disease.

Small, Nonpeptide p75NTR Ligands Induce Survival Signaling and Inhibit proNGF-Induced Death

Studies showing that neurotrophin binding to p75NTR can promote cell survival in the absence of Trk (tropomyosin-related kinase) receptors, together with recent structural data indicating that NGF may bind to p75NTR in a monovalent manner, raise the possibility that small molecule p75NTR ligands that positively regulate survival might be found. A pharmacophore designed to capture selected structural and physical chemical features of a neurotrophin domain known to interact with p75NTR was applied to in silico screening of small molecule libraries. Small, nonpeptide, monomeric compounds were identified that interact with p75NTR. In cells showing trophic responses to neurotrophins, the compounds promoted survival signaling through p75NTR-dependent mechanisms. In cells susceptible to proneurotrophin-induced death, compounds did not induce apoptosis but inhibited proneurotrophin-mediated death. These studies identify a unique range of p75NTR behaviors that can result from isolated receptor liganding and establish several novel therapeutic leads.