Christine M. Gall

A phase 1 clinical trial of nerve growth factor gene therapy for Alzheimer disease

Cholinergic neuron loss is a cardinal feature of Alzheimer disease. Nerve growth factor (NGF) stimulates cholinergic function, improves memory and prevents cholinergic degeneration in animal models of injury, amyloid overexpression and aging. We performed a phase 1 trial of ex vivo NGF gene delivery in eight individuals with mild Alzheimer disease, implanting autologous fibroblasts genetically modified to express human NGF into the forebrain. After mean follow-up of 22 months in six subjects, no long-term adverse effects of NGF occurred. Evaluation of the Mini-Mental Status Examination and Alzheimer Disease Assessment Scale-Cognitive subcomponent suggested improvement in the rate of cognitive decline. Serial PET scans showed significant (P < 0.05) increases in cortical 18-fluorodeoxyglucose after treatment. Brain autopsy from one subject suggested robust growth responses to NGF. Additional clinical trials of NGF for Alzheimer disease are warranted.

BDNF mRNA expression is increased in adult rat forebrain after limbic seizures: temporal patterns of induction distinct from NGF.

We have localized brain-derived neurotrophic factor (BDNF) mRNA in rat brain and examined its regulation by seizure activity. In situ hybridization of BDNF 35S-cRNA most prominently labeled neurons in hippocampal stratum pyramidale and stratum granulosum, superficial olfactory cortex, pyramidal cell layers of neocortex, amygdala, claustrum, endopiriform nucleus, anterior olfactory nucleus, and ventromedial hypothalamus. Hybridization to BDNF mRNA was markedly increased in all of these regions after lesion-induced recurrent limbic seizures and within dentate gyrus granule cells following one electrically stimulated epileptiform afterdischarge. In contrast to seizure-elicited changes in nerve growth factor (NGF) mRNA expression, increases in BDNF mRNA occur in a greater number of different neuronal populations and develop several hours more rapidly in extrahippocampal loci. These results indicate that regulation by physiological activity may be an intrinsic property of this class of neurotrophic factor but that, in the recurrent seizure paradigm, different mechanisms mediate increased expression of mRNAs for BDNF and NGF outside hippocampus.

An anorexic lipid mediator regulated by feeding

Oleylethanolamide (OEA) is a natural analogue of the endogenous cannabinoid anandamide. Like anandamide, OEA is produced in cells in a stimulus-dependent manner and is rapidly eliminated by enzymatic hydrolysis, suggesting a function in cellular signalling. However, OEA does not activate cannabinoid receptors and its biological functions are still unknown. Here we show that, in rats, food deprivation markedly reduces OEA biosynthesis in the small intestine. Administration of OEA causes a potent and persistent decrease in food intake and gain in body mass. This anorexic effect is behaviourally selective and is associated with the discrete activation of brain regions (the paraventricular hypothalamic nucleus and the nucleus of the solitary tract) involved in the control of satiety. OEA does not affect food intake when injected into the brain ventricles, and its anorexic actions are prevented when peripheral sensory fibres are removed by treatment with capsaicin. These results indicate that OEA is a lipid mediator involved in the peripheral regulation of feeding.

BDNF Protein Measured by a Novel Enzyme Immunoassay in Normal Brain and after Seizure: Partial Disagreement with mRNA Levels

Messenger RNA for brain‐derived neurotrophic factor (BDNF) is distributed in many brain regions and regulated by excitatory neuronal activity. Despite numerous studies of BDNF mRNA, the distribution and regulation of BDNF protein are poorly understood because of the difficulty of its quantitative measurement. We have established a two‐site enzyme immunoassay that detects trace amounts of BDNF protein (>1 pg/assay) but not other neurotrophins or growth factors. The highest levels of BDNF in adult rat brain were found in the hippocampus, followed by the hypothalamus, neocortex, cerebellum, thalamus and striatum. This pattern is similar, but not identical, to the distribution of BDNF mRNA. A similar disparity between BDNF protein and mRNA levels was observed in their changes after hilus lesion‐induced limbic seizures. In limbic structures, BDNF concentrations remained elevated 4 days after seizure onset, whereas BDNF mRNA has been reported previously to return to basal levels within 46 h. The temporal and spatial differences between the dynamics of protein and mRNA levels suggest the importance of post‐translational and/or subcellular processes for BDNF production. The persistence of the increases in BDNF content was also reflected in its biological activity, e.g. peptidergic differentiation activity. After limbic seizures, neuropeptide Y content was most markedly and persistently elevated in the entorhinal/amygdaloid region, where the most sustained up‐regulation of BDNF protein was observed. These results suggest that the sustained increase of BDNF protein in these limbic structures is involved in prolonged post‐seizure phenomena, including peptidergic alterations.

BDNF and epilepsy: too much of a good thing?

Various studies have shown that brain-derived neurotrophic factor (BDNF) increases neuronal excitability and is localized and upregulated in areas implicated in epileptogenesis. Seizure activity increases the expression of BDNF mRNA and protein, and recent studies have shown that interfering with BDNF signal transduction inhibits the development of the epileptic state in vivo. These results suggest that BDNF contributes to epileptogenesis. Further analysis of the cellular and molecular mechanisms by which BDNF influences excitability and connectivity in adult brain could provide novel concepts and targets for anticonvulsant or anti-epileptogenic therapy.

KAINIC ACID-INDUCED SEIZURES STIMULATE INCREASED EXPRESSION OF NERVE GROWTH FACTOR MRNA IN RAT HIPPOCAMPUS

The influence of kainic acid (KA)-induced limbic seizure activity on the expression of mRNA for nerve growth factor (NGF) in adult rat brain was studied using in situ hybridization and S1 nuclease protection techniques with RNA probes complementary to murine and rat NGF mRNA. Within hippocampus, intracerebroventricular injection of 0.5 microgram KA caused a dramatic bilateral increase in hybridization of the 35S-labeled cRNA within stratum granulosum. This increase was first evident 1 h post-KA, appeared maximal at approximately 20-fold control levels at 2-3 h post-injection, and declined to control levels by 48 h post-injection. During the period of maximal hybridization, all but the deepest cells within stratum granulosum appeared to be autoradiographically labeled. Hybridization of the NGF cRNA probe was also increased within superficial layers of piriform and entorhinal cortex and, to much lesser extent, within scattered neurons of layers II and III of neocortex in KA-treated rats. In olfactory cortical areas, hybridization was maximally elevated 15.5-24.5 h after KA injection. In contrast to these effects, KA treatment did not consistently influence the density of hybridization, or number of neurons labeled, within the dentate gyrus hilus or the hippocampus proper (CA1-CA3). In agreement with the in situ hybridization results, S1 nuclease protection assay detected KA-induced increases in hybridization within pooled dentate gyrus/CA1 samples, but not hippocampal CA3 samples. These data support the conclusion that seizure activity stimulates a transient increase in NGF expression by select populations of forebrain neurons and indicates that experimental seizure paradigms might be further exploited for analyses of the mechanisms of NGF regulation and processing in the adult brain.

Brain-Derived Neurotrophic Factor Promotes Long-Term Potentiation-Related Cytoskeletal Changes in Adult Hippocampus

Brain-derived neurotrophic factor (BDNF) is an extremely potent, positive modulator of theta burst induced long-term potentiation (LTP) in the adult hippocampus. The present studies tested whether the neurotrophin exerts its effects by facilitating cytoskeletal changes in dendritic spines. BDNF caused no changes in phalloidin labeling of filamentous actin (F-actin) when applied alone to rat hippocampal slices but markedly enhanced the number of densely labeled spines produced by a threshold level of theta burst stimulation. Conversely, the BDNF scavenger TrkB–Fc completely blocked increases in spine F-actin produced by suprathreshold levels of theta stimulation. TrkB–Fc also blocked LTP consolidation when applied 1–2 min, but not 10 min, after theta trains. Additional experiments confirmed that p21 activated kinase and cofilin, two actin-regulatory proteins implicated in spine morphogenesis, are concentrated in spines in mature hippocampus and further showed that both undergo rapid, dose-dependent phosphorylation after infusion of BDNF. These results demonstrate that the influence of BDNF on the actin cytoskeleton is retained into adulthood in which it serves to positively modulate the time-dependent LTP consolidation process.

Integrin Subunit Gene Expression Is Regionally Differentiated in Adult Brain

Integrins are a diverse family of heterodimeric (αβ) adhesion receptors recently shown to be concentrated within synapses and involved in the consolidation of long-term potentiation. Whether neuronal types or anatomical systems in the adult rat brain are coded by integrin type was studied in the present experiments by mapping the relative densities of mRNAs for nine α and four β subunits. Expression patterns were markedly different and in some regions complementary. General results and areas of notable labeling were as follows: α1—limited neuronal expression, neocortical layer V, hippocampal CA3; α3 and α5—diffuse neuronal and glial labeling, Purkinje cells, hippocampal stratum pyramidale, locus coeruleus (α3); α4— discrete limbic regions, olfactory cortical layer II, hippocampal CA2; α6—most prominently neuronal, neocortical subplate, endopiriform, subiculum; α7—discrete, all neocortical layers, hippocampal granule cells and CA3, cerebellar granule and Purkinje cells, all efferent cranial nerve nuclei; α8—discrete neuronal, deep cortex, hippocampal CA1, basolateral amygdala, striatum; αV—all cortical layers, striatum, Purkinje cells; β4—dentate gyrus granule cells; β5—broadly distributed, neocortex, medial amygdala, cerebellar granule and Purkinje cells, efferent cranial nerve nuclei; α2, β2, and β3—mRNAs not detected. These results establish that brain subfields express different balances of integrin subunits and thus different integrin receptors. Such variations will determine which matrix proteins are recognized by neurons and the types of intraneuronal signaling generated by matrix binding. They also could generate important differences in synaptic plasticity across brain systems.

Changes in Synaptic Morphology Accompany Actin Signaling during LTP

Stabilization of long-term potentiation (LTP) is commonly proposed to involve changes in synaptic morphology and reorganization of the spine cytoskeleton. Here we tested whether, as predicted from this hypothesis, induction of LTP by theta-burst stimulation activates an actin regulatory pathway and alters synapse morphology within the same dendritic spines. TBS increased severalfold the numbers of spines containing phosphorylated (p) p21-activated kinase (PAK) or its downstream target cofilin; the latter regulates actin filament assembly. The PAK/cofilin phosphoproteins were increased at 2 min but not 30 s post-TBS, peaked at 7 min, and then declined. Double immunostaining for the postsynaptic density protein PSD95 revealed that spines with high pPAK or pCofilin levels had larger synapses (+60–70%) with a more normal size frequency distribution than did neighboring spines. Based on these results and simulations of shape changes to synapse-like objects, we propose that theta stimulation markedly increases the probability that a spine will enter a state characterized by a large, ovoid synapse and that this morphology is important for expression and later stabilization of LTP.

Hippocampal Dysfunction and Cognitive Impairments Provoked by Chronic Early-Life Stress Involve Excessive Activation of CRH Receptors

Chronic stress impairs learning and memory in humans and rodents and disrupts long-term potentiation (LTP) in animal models. These effects are associated with structural changes in hippocampal neurons, including reduced dendritic arborization. Unlike the generally reversible effects of chronic stress on adult rat hippocampus, we have previously found that the effects of early-life stress endure and worsen during adulthood, yet the mechanisms for these clinically important sequelae are poorly understood. Stress promotes secretion of the neuropeptide corticotropin-releasing hormone (CRH) from hippocampal interneurons, activating receptors (CRF1) located on pyramidal cell dendrites. Additionally, chronic CRF1 occupancy negatively affects dendritic arborization in mouse organotypic slice cultures, similar to the pattern observed in middle-aged, early-stressed (CES) rats. Here we found that CRH expression is augmented in hippocampus of middle-aged CES rats, and then tested whether the morphological defects and poor memory performance in these animals involve excessive activation of CRF1 receptors. Central or peripheral administration of a CRF1 blocker following the stress period improved memory performance of CES rats in novel-object recognition tests and in the Morris water maze. Consonant with these effects, the antagonist also prevented dendritic atrophy and LTP attenuation in CA1 Schaffer collateral synapses. Together, these data suggest that persistently elevated hippocampal CRH–CRF1 interaction contributes importantly to the structural and cognitive impairments associated with early-life stress. Reducing CRF1 occupancy post hoc normalized hippocampal function during middle age, thus offering potential mechanism-based therapeutic interventions for children affected by chronic stress.