J. Regino Perez-Polo

The regulation of amyloid precursor protein metabolism by cholinergic mechanisms and neurotrophin receptor signaling

The increased expression and/or abnormal processing of the amyloid precursor protein (APP) is associated with the formation of amyloid plaques and cerebrovascular amyloid deposits, which are one of the major morphological hallmarks of Alzheimers disease (AD). Among the processes regulating APP metabolism, the proteolytic cleavage of APP into amyloidogenic or nonamyloidogenic fragments is of special interest. The cleavage of the APP by the alpha-secretase within the beta-amyloid sequence generates nonamyloidogenic C-terminal APP fragments and soluble APPs alpha, which has neurotrophic and neuroprotective activities. Proteolytic processing of APP by beta-secretase, on the other hand, exposes the N-terminus of beta-amyloid, which is liberated after gamma-secretase cleavage at the variable amyloid C-terminus. The resulting 39-43 amino acid beta-amyloid may be neurotoxic and disrupt neuronal connectivity after its accumulation in senile plaques. In this review, we discuss evidence derived from in vitro experiments, suggesting that the stimulation of protein kinase C (PKC)-coupled M1/M3 muscarinic acetylcholine receptors increases the nonamyloidogenic, secretory pathway of APP processing. It has also been shown in animal models that under conditions of reduced M1/M3 muscarinic acetylcholine receptor stimulation the secretory pathway of APP processing is inhibited and that constitutive upregulation of M1/M3-associated PKC increases APP secretion. Thus, the cortical cholinergic hypoactivity characteristic of AD may inhibit the nonamyloidogenic APP processing pathway and lead to increased beta-amyloid generation. It has been shown in vitro that nerve growth factor (NGF)-associated signaling also influences the expression and catabolism of APP. Recent experiments with NGF-responsive cells revealed a specific role for the high-affinity NGF receptor, TrkA, in the increases in secretory APP processing and a role for the low-affinity neurotrophin receptor, p75NTR, in the transcriptional regulation of APP. Therefore, treatments with NGF could ameliorate cortical cholinergic dysfunction in AD. These findings may influence the design of therapeutic strategies aimed at stimulating cholinergic function and at increasing nonamyloidogenic APP processing without elevating APP expression.

Inhibition of nuclear factor kappa B (NFκB) activity induces nerve growth factor-resistant apoptosis in PC12 cells

The mechanism(s) underlying nerve growth factor (NGF)‐mediated rescue of neurons from apoptosis is poorly understood, although it is well established that the high‐affinity NGF receptor (TrkA) plays a pivotal role in mediating NGF effects. The report that the low‐affinity NGF receptor (p75NGFR) can induce apoptosis prompted us to analyze the role played by a putative p75NGFR‐associated signal‐transduction element, the transcription factor nuclear factor kappa B (NFκB), in the modulation of apoptosis in PC12 cells. Here, we report that inhibition of NFκB function results in apoptosis of rat PC12 cells, a neuroblast‐like cell line model of NGF‐responsive neural tissues. Furthermore, NGF did not protect PC12 cells from cell death induced by the inhibition of NFκB. These results indicate that NFκB function is essential to maintain PC12 cell survival and to permit NGF‐mediated rescue, consistent with the idea that signaling elements potentially associated with both TrkA‐ and p75NGFR are involved in the regulation of apoptosis. J. Neurosci. Res. 47:155–162, 1997.

Protective effects of bone marrow stromal cell transplantation in injured rodent brain: synthesis of neurotrophic factors.

Several groups have suggested that transplantation of marrow stromal cells (MSCs) promotes functional recovery in animal models of brain trauma. Recent studies indicate that tissue replacement by this method may not be the main source of therapeutic benefit, as transplanted MSCs have only limited ability to replace injured central nervous system (CNS) tissue. To gain insight into the mechanisms responsible for such effects, we systematically investigated the therapeutic potential of MSCs for treatment of brain injury. Using in vitro studies, we detected the synthesis of various growth factors, including nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF), glial cell line‐derived neurotrophic factor (GDNF), and neurotrophin‐3 (NT‐3). Enzyme‐linked immunosorbent assay (ELISA) demonstrated that MSCs cultured in Dulbeccos modified Eagle medium (DMEM) produced substantial amounts of NGF for at least 7 weeks, whereas the levels of BDNF, GDNF and NT‐3 remained unchanged. In studies in mice, after intraventricular injection of MSCs, NGF levels were increased significantly in cerebrospinal fluid by ELISA, confirming our cell culture results. Further studies showed that treatment of traumatic brain injury with MSCs could attenuate the loss of cholinergic neuronal immunostaining in the medial septum of mice. These studies demonstrate for the first time that by increasing the brain concentration of NGF, intraventricularly transplanted MSCs might play an important role in the treatment of traumatic brain injury.

Differential regulation of BACE1 promoter activity by nuclear factor-κB in neurons and glia upon exposure to β-amyloid peptides

The brains of Alzheimers disease (AD) patients display cerebrovascular and parenchymal deposits of β‐amyloid (Aβ) peptides, which are derived by proteolytic processing by the β‐site APP‐cleaving enzyme 1 (BACE1) of the amyloid precursor protein (APP). The rat BACE1 promoter has a nuclear factor‐κB (NF‐κB) binding site. Deletion studies with a BACE1 promoter/luciferase reporter suggest that the NF‐κB binding DNA consensus sequence plays a suppressor role, when occupied by NF‐κB, in the regulation of neuronal brain BACE1 expression. Here we characterize a signal transduction pathway that may be responsible for the increases in Aβ associated with AD. We propose that the transcription factor NF‐κB acts as a repressor in neurons but as an activator of BACE1 transcription in activated astrocytes present in the CNS under chronic stress, a feature present in the AD brain. The activated astrocytic stimulation of BACE1 may in part account for increased BACE1 transcription and subsequent processing of Aβ in a cell‐specific manner in the aged and AD brain. As measured by reporter gene promoter constructs and endogenous BACE1 protein expression, a functional NF‐κB site was stimulatory in activated astrocytes and Aβ‐exposed neuronal cells and repressive in neuronal and nonactivated astrocytic cells. Given the evidence for increased levels of activated astrocytes in the aged brain, the age‐ and AD‐associated increases in NF‐κB in brain may be significant contributors to increases in Aβ, acting as a positive feedback loop of chronic inflammation, astrocyte activation, increased p65/p50 activation of BACE1 transcription, and further inflammation.

Effects of Nerve Growth Factor on Glutathione Peroxidase and Catalase in PC 12 Cells

Abstract: Nerve growth factor (NGF) is a member of the neuro‐ trophin family and is required for the survival and maintenance of peripheral sympathetic and sensory ganglia. In the CNS, NGF regulates cholinergic expression by basal forebrain cholinergic neurons. NGF also stimulates cellular resistance to oxidative stress in the PC12 cell line and protects PC12 cells from the toxic effects of reactive oxygen species. The hypothesis that NGF protection involves changes in antioxidant enzyme expression was tested by measuring its effects on catalase and glutathione per‐ oxidase (GSH Px) mRNA expression in PC12 cells. NGF increased catalase and GSH Px mRNA levels in PC 12 cells in a time‐ and dose‐dependent manner. There was also a corresponding increase in the enzyme activities of catalase and GSH Px. Thus, NGF can provide cytoprotection to PC12 cells by inducing the free radical scavenging enzymes catalase and GSH Px.

Transcriptional profiling of spinal cord injury‐induced central neuropathic pain

Central neuropathic pain (CNP) is an important problem following spinal cord injury (SCI), because it severely affects the quality of life of SCI patients. As in the patient population, the majority of rats develop significant allodynia (CNP rats) after moderate SCI. However, about 10% of SCI rats do not develop allodynia, or develop significantly less allodynia than CNP rats (non‐CNP rats). To identify transcriptional changes underlying CNP development after SCI, we used Affymetrix DNA microarrays and RNAs extracted from the spinal cords of CNP and non‐CNP rats. DNA microarry analysis showed significantly increased expression of a number of genes associated with inflammation and astrocytic activation in the spinal cords of rats that developed CNP. For example, mRNA levels of glial fibrilary acidic protein (GFAP) and Aquaporin 4 (AQP4) significantly increased in CNP rats. We also found that GFAP, S100β and AQP4 protein elevation persisted for at least 9 months throughout contused spinal cords, consistent with the chronic nature of CNP. Thus, we hypothesize that CNP development results, in part, from dysfunctional, chronically “over‐activated” astrocytes. Although, it has been shown that activated astrocytes are associated with peripheral neuropathic pain, this has not previously been demonstrated in CNP after SCI.

Alzheimer's disease β-secretase BACE1 is not a neuron-specific enzyme

The brains of Alzheimers disease (AD) patients are morphologically characterized by neurofibrillar abnormalities and by parenchymal and cerebrovascular deposits of β‐amyloid peptides. The generation of β‐amyloid peptides by proteolytical processing of the amyloid precursor protein (APP) requires the enzymatic activity of the β‐site APP cleaving enzyme 1 (BACE1). The expression of this enzyme has been localized to the brain, in particular to neurons, indicating that neurons are the major source of β‐amyloid peptides in brain. Astrocytes, on the contrary, are known to be important for β‐amyloid clearance and degradation, for providing trophic support to neurons, and for forming a protective barrier between β‐amyloid deposits and neurons. However, under certain conditions related to chronic stress, the role of astrocytes may not be beneficial. Here we present evidence demonstrating that astrocytes are an alternative source of BACE1 and therefore may contribute to β‐amyloid plaque formation. While resting astroyctes in brain do not express BACE1 at detectable levels, cultured astrocytes display BACE1 promoter activity and express BACE1 mRNA and enzymatically active BACE1 protein. Additionally, in animal models of chronic gliosis and in brains of AD patients, there is BACE1 expression in reactive astrocytes. This would suggest that the mechanism for astrocyte activation plays a role in the development of AD and that therapeutic strategies that target astrocyte activation in brain may be beneficial for the treatment of AD. Also, there are differences in responses to chronic versus acute stress, suggesting that one consequence of chronic stress is an incremental shift to different phenotypic cellular states.

Neuronal and glial β-secretase (BACE) protein expression in transgenic Tg2576 mice with amyloid plaque pathology

We measured tissue distribution and expression pattern of the beta‐site amyloid precursor protein (APP)‐cleaving enzyme (BACE) in the brains of transgenic Tg2576 mice that show amyloid pathology. BACE protein was expressed at high levels in brain; at lower levels in heart and liver; and at very low levels in pancreas, kidney, and thymus and was almost absent in spleen and lung when assayed by Western blot analysis. We observed strictly neuronal expression of BACE protein in the brains of nontransgenic control mice, with the most robust immunocytochemical labeling present in the cerebral cortex, hippocampal formation, thalamus, and cholinergic basal forebrain nuclei. BACE protein levels did not differ significantly between control and transgenic mice or as a result of aging. However, in the aged, 17‐month‐old Tg2576 mice there was robust amyloid plaque formation, and BACE protein was also present in reactive astrocytes present near amyloid plaques, as shown by double immunofluorescent labeling and confocal laser scanning microscopy. The lack of astrocytic BACE immunoreactivity in young transgenic Tg2576 mice suggests that it is not the APP overexpression but rather the amyloid plaque formation that stimulates astrocytic BACE expression in Tg2576 mice. Our data also suggest that the neuronal overexpression of APP does not induce the overexpression of its metabolizing enzyme in neurons. Alternatively, the age‐dependent accumulation of amyloid plaques in the Tg2576 mice does not require increased neuronal expression of BACE. Our data support the hypothesis that neurons are the primary source of β‐amyloid peptides in brain and that astrocytic β‐amyloid generation may contribute to amyloid plaque formation at later stages or under conditions when astrocytes are activated. J. Neurosci. Res. 64:437–446, 2001.

Participation of active oxygen species in 6-hydroxydopamine toxicity to a human neuroblastoma cell line

Catalase, superoxide dismutase, and dimethylsulfoxide were tested for their ability to prevent the cytotoxic effect of 6-hydroxydopamine (6-OHDA) on the human neuroblastoma line SY5Y. Viability was measured at two time points after 6-OHDA treatment: at 3 hr by means of amino acid incorporation and at 24 hr by trypan blue dye exclusion. Survival of cells treated concomitantly with catalase (50 microgram/ml) and 6-OHDA was at least 90 per cent that of untreated controls. Cells receiving 6-OHDA alone showed less than 30 per cent survival relative to untreated controls. Superoxide dismutase (50 microgram/ml) temporarily protected cells from a high concentration of 60-OHDA. Dimethylsulfoxide treatment increased survival from the control level 24 hr after treatment with 6-OHDA. Two other cell lines (A1B1 human glial cells and CHO fibroblasts) had intermediate and high resistance to the drug, respectively, compared to the low resistance of SY5Y cells. CHO and SY5Y cells had similar responses to 6-OHDA and to H2O2 when tested at twice the molarity of 6-OHDA. Specific activities of three enzymes known to detoxify H2O2 or H2O2-generated organic hydroperoxides (catalase, glutathione S-transferase, and glutathione peroxidase) were compared in the three cell lines. Catalase activity was 2.5 times as high as in A1B1 and CHO cells as in SY5Y cells when expressed as units/mg protein and 7 times as high in units/culture dish. Other enzyme activities showed no correlation to 6-OHDA resistance.

Poly(ADP-ribose) polymerase inhibition prevents both apoptotic-like delayed neuronal death and necrosis after H2O2 injury

Toxic reactive oxygen species (ROS) such as hydrogen peroxide, nitric oxide, superoxide, and the hydroxyl radical are generated in a variety of neuropathological conditions and cause significant DNA damage. We determined the effects of 3‐aminobenzamide (AB), an inhibitor of the DNA repair enzyme poly(ADP‐ribose) polymerase (PARP), on cell death in differentiated PC12 cells, a model of sympathetic neurons, after H2O2 injury. Exposure to 0.5 mm H2O2 resulted in a significant decrease in intracellular NAD(H), NADP(H), and ATP levels. This injury resulted in the death of 90% of the cells with significant necrosis early (2 h) after injury and increased apoptosis (12–24 h after injury), as measured by PS exposure and the presence of cytoplasmic oligonucleosomal fragments. Treatment with 2.5 mm AB restored pyridine nucleotide and ATP levels and ameliorated cell death (65% versus 90%) by decreasing the extent of both necrosis and apoptosis. Interestingly, we observed that H2O2‐induced injury caused a delayed cell death exhibiting features of apoptosis but in which caspase‐3 like activity was absent. Moreover, pretreatment with AB restored caspase‐3‐like activity. Our results suggest that apoptosis and necrosis are both triggered by PARP overactivation, and that maintenance of cellular energy levels after injury by inhibiting PARP shifts cell death from necrosis to apoptosis.