H. Uri Saragovi

Loss of nucleus basalis neurons containing trkA immunoreactivity in individuals with mild cognitive impairment and early Alzheimer's disease

Recent studies indicate that there is a marked reduction in trkA–containing nucleus basalis neurons in end–stage Alzheimers disease (AD). We used unbiased stereological counting procedures to determine whether these changes extend to individuals with mild cognitive impairment (MCI) without dementia from a cohort of people enrolled in the Religious Orders Study. Thirty people (average age 84.7 years) came to autopsy. All individuals were cognitively tested within 12 months of death (average MMSE 24.2). Clinically, 9 had no cognitive impairment (NCI), 12 were categorized with MCI, and 9 had probable AD The average number of trkA–immunoreactive neurons in persons with NCI was 196, 632 ± 12,093 (n = 9), for those with MCI it was 106,110 ± 14,565, and for those with AD it was 86,978 ± 12,141. Multiple comparisons showed that both those with MCI and those with AD had significant loss in the number of trkA–containing neurons compared to those with NCI (46% decrease for MCI, 56% for AD). An analysis of variance revealed that the total number of neurons containing trkA immunoreactivity was related to diagnostic classification (P < 0.001), with a significant reduction in AD and MCI compared to NCI but without a significant difference between MCI and AD. Cell density was similarly related to diagnostic classification (P < 0.001). There was a significant correlation with the Boston Naming Test and with a global score measure of cognitive function. The number of trkA–immunoreactive neurons was not correlated with MMSE, age at death, education, apolipoprotein E allele status, gender, or Braak score. These data indicate that alterations in the number of nucleus basalis neurons containing trkA immunoreactivity occurs early and are not accelerated from the transition from MCI to mild AD. J. Comp. Neurol. 427:19–30, 2000.

Reduction in p140-TrkA receptor protein within the nucleus basalis and cortex in Alzheimer's disease.

It has been hypothesized that the diminished transport of nerve growth factor (NGF) seen within cholinergic basal forebrain (CBF) neurons in Alzheimers disease (AD) results from a defect in the expression of its high-affinity trkA receptor. The present study used an anti-human trkA-specific monoclonal antibody (mAb 5C3) that recognizes the NGF docking site, combined with quantitative optical densitometry, to evaluate whether expression of the trkA protein is altered within the nucleus basalis and its cortical projection sites in AD. In normal aged humans, trkA immunoreactivity revealed a continuum of positive neurons extending throughout all CBF subfields. In addition, trkA-positive neurons were scattered throughout the olfactory tubercle and striatum. These regions also displayed intense trkA neuropil staining. Although fewer in total number, remaining CBF perikarya in AD displayed a significant decrease in trkA levels relative to aged controls. Biochemical analysis revealed a significant reduction in trkA protein within both the nucleus basalis and the frontal cortex in AD relative to aged controls. In contrast, trkA levels in the caudate nucleus were unaffected. The decrease in trkA protein in conjunction with our recent observations that the message for trkA is reduced within individual CBF neurons in AD supports the concept that defects in the production and/or utilization of the trkA receptor may be a key event mediating degeneration of NGF-responsive CBF neurons in this disease.

Reduction of cortical TrkA but not p75NTR protein in early‐stage Alzheimer's disease

Degeneration of cholinergic nucleus basalis (NB) cortical projection neurons is associated with cognitive decline in late‐stage Alzheimers disease (AD). NB neuron survival is dependent on coexpression of the nerve growth factor (NGF) receptors p75NTR and TrkA, which bind NGF in cortical projection sites. We have shown previously a significant reduction of NB perikarya expressing p75NTR and TrkA protein during the early stages of AD. Whether there is a concomitant reduction in cortical levels of these receptors during the progression of AD is unknown. p75NTR and TrkA protein was evaluated by quantitative immunoblotting in five cortical regions (anterior cingulate, superior frontal, superior temporal, inferior parietal, and visual cortex) of individuals clinically diagnosed with no cognitive impairment (NCI), mild cognitive impairment (MCI), mild/moderate AD, or severe AD. Cortical p75NTR levels were stable across the diagnostic groups. In contrast, TrkA levels were reduced approximately 50% in mild/moderate and severe AD compared with NCI and MCI in all regions except visual cortex. Mini‐Mental Status Examination scores correlated with TrkA levels in anterior cingulate, superior frontal, and superior temporal cortex. The selective reduction of cortical TrkA levels relative to p75NTR may have important consequences for cholinergic NB function during the transition from MCI to AD. Ann Neurol 2004

Development of pharmacological agents for targeting neurotrophins and their receptors

Neurotrophins comprise a family of protein growth factors that control the survival, growth, and/or differentiation of neurons and several other cell populations derived from the neuroectoderm. Neurotrophins and their receptors are important targets for the therapy of human disease, with potential applications ranging from the treatment of chronic or acute neurodegeneration to pain and cancer. Neurotrophins have been used clinically but are poor pharmacological agents. Consequently, approaches to develop pharmacological agents that target neurotrophins, their receptors or neurotrophin signaling pathways have been attempted.

Inhibition of p75NTR in glia potentiates TrkA-mediated survival of injured retinal ganglion cells

Little is known about the molecular mechanisms that limit the ability of retinal neurons to respond to neurotrophic factor stimulation following axonal injury. In the adult retina, nerve growth factor (NGF) binds to TrkA (expressed by neurons) and p75(NTR) (expressed by Müller glia), but fails to promote the survival of axotomized retinal ganglion cells (RGCs). We addressed the functional role of TrkA and p75(NTR) in this lack of survival by using peptidomimetic agonistic or antagonistic ligands specific for each receptor. While administration of exogenous NGF failed to rescue axotomized RGCs, administration of selective TrkA agonists led to robust neuroprotection. Surprisingly, we found a remarkable survival of axotomized RGCs following pharmacological inhibition of p75(NTR) or in p75(NTR) knockout mice. Combination of NGF or TrkA agonists with p75(NTR) antagonists further potentiated RGC neuroprotection in vivo, an effect that was greater than each treatment alone. NGF can therefore be neuroprotective when acting on neuronal TrkA receptors but engagement of p75(NTR) on glial cells antagonizes this effect. Our data reveal a novel mechanism by which p75(NTR) expressed on retinal glia can profoundly influence neuronal survival.

Long-Lasting Rescue of Age-Associated Deficits in Cognition and the CNS Cholinergic Phenotype by a Partial Agonist Peptidomimetic Ligand of TrkA

Previously, we developed a proteolytically stable small molecule peptidomimetic termed D3 as a selective ligand of the extracellular domain of the TrkA receptor for the NGF. Ex vivo D3 was defined as a selective, partial TrkA agonist. Here, the in vivo efficacy of D3 as a potential therapeutic for cholinergic neurons was tested in cognitively impaired aged rats, and we compared the consequence of partial TrkA activation (D3) versus full TrkA/p75 activation (NGF). We show that in vivo D3 binds to TrkA receptors and affords a significant and long-lived phenotypic rescue of the cholinergic phenotype both in the cortex and in the nucleus basalis. The cholinergic rescue was selective and correlates with a significant improvement of memory/learning in cognitively impaired aged rats. The effects of the synthetic ligand D3 and the natural ligand NGF were comparable. Small, proteolytically stable ligands with selective agonistic activity at a growth factor receptor may have therapeutic potential for neurodegenerative disorders.

A kinase-deficient TrkC receptor isoform activates Arf6–Rac1 signaling through the scaffold protein tamalin

Neurotrophins play an essential role in mammalian development. Most of their functions have been attributed to activation of the kinase-active Trk receptors and the p75 neurotrophin receptor. Truncated Trk receptor isoforms lacking the kinase domain are abundantly expressed during development and in the adult; however, their function and signaling capacity is largely unknown. We show that the neurotrophin-3 (NT3) TrkCT1-truncated receptor binds to the scaffold protein tamalin in a ligand-dependent manner. Moreover, NT3 initiation of this complex leads to activation of the Rac1 GTPase through adenosine diphosphate-ribosylation factor 6 (Arf6). At the cellular level, NT3 binding to TrkCT1–tamalin induces Arf6 translocation to the membrane, which in turn causes membrane ruffling and the formation of cellular protrusions. Thus, our data identify a new signaling pathway elicited by the kinase-deficient TrkCT1 receptor. Moreover, we establish NT3 as an upstream regulator of Arf6.

Ligand activation of nerve growth factor receptor TrkA protects monocytes from apoptosis

Nerve growth factor (NGF) receptors are expressed in different cell types outside the nervous system, and increasing evidence indicates that NGF can act as a regulatory molecule during inflammatory and immune responses. In this study, we show that triggering of the high‐affinity NGF receptor TrkA with agonists protects monocytes from apoptosis induced by gliotoxin or UVB radiation. TrkA stimulation up‐regulates the expression of the anti‐apoptotic Bcl‐2 family members, Bcl‐2, Bcl‐XL, and Bfl‐1. On the other hand, TrkA stimulation does not change the expression of MHC, CD80, CD86, CD40, and CD54 molecules, nor the antigen‐presenting function of monocytes. In addition, during in vitro monocyte to dendritic cell differentiation TrkA expression is progressively lost, suggesting that NGF selectively affects monocyte but not dendritic cell survival.

Chronic and acute models of retinal neurodegeneration TrkA activity are neuroprotective whereas p75NTR activity is neurotoxic through a paracrine mechanism.

In normal adult retinas, NGF receptor TrkA is expressed in retinal ganglion cells (RGC), whereas glia express p75NTR. During retinal injury, endogenous NGF, TrkA, and p75NTR are up-regulated. Paradoxically, neither endogenous NGF nor exogenous administration of wild type NGF can protect degenerating RGCs, even when administered at high frequency. Here we elucidate the relative contribution of NGF and each of its receptors to RGC degeneration in vivo. During retinal degeneration due to glaucoma or optic nerve transection, treatment with a mutant NGF that only activates TrkA, or with a biological response modifier that prevents endogenous NGF and pro-NGF from binding to p75NTR affords significant neuroprotection. Treatment of normal eyes with an NGF mutant-selective p75NTR agonist causes progressive RGC death, and in injured eyes it accelerates RGC death. The mechanism of p75NTR action during retinal degeneration due to glaucoma is paracrine, by increasing production of neurotoxic proteins TNF-α and α2-macroglobulin. Antagonists of p75NTR inhibit TNF-α and α2-macroglobulin up-regulation during disease, and afford neuroprotection. These data reveal a balance of neuroprotective and neurotoxic mechanisms in normal and diseased retinas, and validate each neurotrophin receptor as a pharmacological target for neuroprotection.

An agonistic TrkB mAb causes sustained TrkB activation, delays RGC death, and protects the retinal structure in optic nerve axotomy and in glaucoma.

PURPOSE Brain-derived neurotrophic factor (BDNF) receptors TrkB and p75(NTR) are expressed in the retina. However, exogenous BDNF does not provide retinal ganglion cells (RGCs) with long-lasting neuroprotection in vivo during optic nerve axotomy or in glaucoma rat models of neurodegeneration. The authors set out to answer the hypothesis that a selective TrkB agonist might afford more efficient neuroprotection. METHODS Animal models of acute neurodegeneration (complete optic nerve axotomy) and chronic neurodegeneration (ocular hypertension, glaucoma) were used. After intravitreal delivery of test agents or controls, surviving RGCs were quantified. Transient or sustained activation of TrkB receptors in vivo was quantified by Western blot analysis retinal samples for TrkB-phosphotyrosine. Time-dependent changes to the neuronal retinal layers were quantified longitudinally by Fourier domain-optical coherence tomography. RESULTS The authors show that a selective TrkB agonist caused long-lived TrkB activation and significantly delayed RGC death in these models of acute and chronic retinal injury in vivo. Importantly, using noninvasive retinal imaging, they also show that a selective TrkB agonist caused preservation of the retinal structure in both animal models, with maintenance of the layers comprising neurons and neuronal fibers. CONCLUSIONS In animal models of acute and chronic neurodegeneration, a TrkB agonist affords long-lasting neuroprotection by causing sustained TrkB activation. The use of structural end points could have prognostic value to evaluate neuroprotection. This work contributes to the understanding of neurotrophic mechanisms underlying RGC death in glaucoma and optic nerve axotomy.