Knockdown of Amyloid Precursor Protein Increases Ion Channel Expression and Alters Ca2+ Signaling Pathways.

Although the physiological role of the full-length Amyloid Precursor Protein (APP) and its proteolytic fragments remains unclear, they are definitively crucial for normal synaptic function. Herein, we report that the downregulation of APP in SH-SY5Y cells, using short hairpin RNA (shRNA), alters the expression pattern of several ion channels and signaling proteins that are involved in synaptic and Ca2+ signaling. Specifically, the levels of GluR2 and GluR4 subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors (AMPAR) were significantly increased with APP knockdown. Similarly, the expression of the majority of endoplasmic reticulum (ER) residing proteins, such as the ER Ca2+ channels IP3R (Inositol 1,4,5-triphosphate Receptor) and RyR (Ryanodine Receptor), the Ca2+ pump SERCA2 (Sarco/endoplasmic reticulum Ca2+ ATPase 2) and the ER Ca2+ sensor STIM1 (Stromal Interaction Molecule 1) was upregulated. A shift towards the upregulation of p-AKT, p-PP2A, and p-CaMKIV and the downregulation of p-GSK, p-ERK1/2, p-CaMKII, and p-CREB was observed, interconnecting Ca2+ signal transduction from the plasma membrane and ER to the nucleus. Interestingly, we detected reduced responses to several physiological stimuli, with the most prominent being the ineffectiveness of SH-SY5Y/APP- cells to mobilize Ca2+ from the ER upon carbachol-induced Ca2+ release through IP3Rs and RyRs. Our data further support an emerging yet perplexing role of APP within a functional molecular network of membrane and cytoplasmic proteins implicated in Ca2+ signaling.

Amyloid-ß Protein Precursor Regulates Depolarization-Induced Calcium-Mediated Synaptic Signaling in Brain Slices.

BACKGROUND: Coordinated calcium influx upon neuronal depolarization activates pathways that phosphorylate CaMKII, ERKs, and the transcription factor CREB and, therefore, expression of pro-survival and neuroprotective genes. Recent evidence indicates that amyloid-ß protein precursor (AßPP) is trafficked to synapses and promotes their formation. At the synapse, AßPP interacts with synaptic proteins involved in vesicle exocytosis and affects calcium channel function. OBJECTIVE: Herein, we examined the role of AßPP in depolarization-induced calcium-mediated signaling using acute cerebral slices from wild-type C57bl/6 mice and AßPP-/- C57bl/6 mice. METHODS: Depolarization of acute cerebral slices from wild-type C57bl/6 and AßPP-/- C57bl/6 mice was used to induce synaptic signaling. Protein levels were examined by western blot and calcium dynamics were assessed using primary neuronal cultures. RESULTS: In the absence of AßPP, decreased pCaMKII and pERKs levels were observed. This decrease was sensitive to the inhibition of N- and P/Q-type Voltage Gated Calcium Channels (N- and P/Q-VGCCs) by ω-conotoxin GVIA and ω-conotoxin MVIIC, respectively, but not to inhibition of L-type VGCCs by nifedipine. However, the absence of AßPP did not result in a statistically significant decrease of pCREB, which is a known substrate of pERKs. Finally, using calcium imaging, we found that down regulation of AßPP in cortical neurons results in a decreased response to depolarization and altered kinetics of calcium response. CONCLUSION: AßPP regulates synaptic activity-mediated neuronal signaling by affecting N- and P/Q-VGCCs.

Investigation of genetic base in the treatment of age-related macular degeneration.

PURPOSE: To determine whether gene polymorphisms which are associated with age-related macular degeneration (AMD) influence treatments' response and specifically the antioxidant supplementation in dry AMD patients, as well as the anti-vascular endothelial growth factor (anti-VEGF) therapy in neovascular AMD patients. METHODS: A total of 170 patients with dry AMD and 52 neovascular AMD patients were genotyped for the following single nucleotide polymorphisms (SNPs): rs1061170/Y402H in CFH gene, rs10490924/A69S in ARMS2 gene, rs9332739/E318D and rs547154/IVS10 in C2 gene, and rs4151667/L9H and rs2072633/IVS17 in CFB gene. Treatment response was evaluated by comparing visual acuity and optical coherence tomography between baseline and at the end of the treatment. RESULTS: Τhe CFH/Y402H variant was associated with the response to antioxidants in dry AMD patients. Carriers of one or two CFH risk alleles displayed a lower chance of responding compared to those with no risk allele. No association of antioxidants' response and ARMS2/A69S genotype was identified. The analysis of the C2 and CFB genetic variants (protective SNPs) revealed that antioxidant supplementation was much more effective in protective SNP carriers. In neovascular AMD patients, the analysis indicated that Y402H homozygous patients were less likely to respond to anti-VEGF therapy compared to heterozygous. Regarding the ARMS2/A69S genotype, carriers of the risk variant experienced significantly worse treatment outcome compared to wild-type patients. CONCLUSION: In AMD patients, the efficacy of the antioxidant supplementation and the anti-VEGF therapy appears to differ by genotype. The detection of genetic variants, associated with treatment responsiveness, could lead to improved visual outcomes through genotype-directed therapy.

Design and synthesis of gallocyanine inhibitors of DKK1/LRP6 interactions for treatment of Alzheimer's disease.

Based on NCI8642, a series of gallocyanine derivatives was synthesized with modifications of the substituent groups in position 1, 2 and 4 of the phenoxazinone scaffold. The effectiveness of gallocyanines to inhibit DKK1/LRP6 interactions and Tau phosphorylation induced by prostaglandin J2 and DKK1 was elucidated by both experimental data and molecular docking simulations. Bis-alkylated with flexible alkyl ester groups on C1 and bis-benzyl gallocyanines provided the most active inhibitors, while amino derivatives on C2 of NCI8642 that have alkoxy or benzyloxy substituents on C4, were less active. Furthermore, it is shown that treating of SHSY5Y cells with NCI8642 derivatives activates Wnt signaling and increases the levels of pGSK3ß kinase and ß-catenin.

Publisher Correction: An integrated bacterial system for the discovery of chemical rescuers of disease-associated protein misfolding.

In the version of this Article originally published, in Fig. 1c-e, on the x axes, the lines labelled 'Aß42' and 'Aß42(F19S;L34P)' grouped the data incorrectly; the line labelled Aß42 should have grouped the data for Random 1-2 and Clones 1-10, and the line labelled Aß42(F19S;L34P) should have only grouped the data for Random 1-2 on the right end of the plots and blots. These figures have now been corrected in all versions of the Article.

Oxygen and Glucose Deprivation Alter Synaptic Distribution of Tau Protein: The Role of Phosphorylation.

Alterations in tau synaptic distribution are considered to underlie synaptic dysfunction observed in Alzheimer's disease (AD). In the present study, brain blood hypoperfusion was simulated in mouse brain slices, and tau levels and phosphorylation were investigated in total extracts, as well as in postsynaptic density fractions (PSDs) and non-PSDs obtained through differential extraction and centrifugation. Oxygen deprivation (OD) resulted in tau dephosphorylation at several AD-related residues and activation of GSK3ß and phosphatase PP2A. On the contrary, glucose deprivation (GD) did not affect total levels of cellular tau or its phosphorylation despite inactivation of GSK3ß. However, tau distribution in PSD and non-PSD fractions and the pattern of tau phosphorylation in these compartments is highly complex. In PSDs, tau was increased under GD conditions and decreased under OD conditions. GD resulted in tau dephosphorylation at Ser199, Ser262, and Ser396 while OD resulted in tau hyperphosphorylation at Ser199 and Ser404. In the non-PSD fraction, GD or OD resulted in lower levels of tau, but the phosphorylation status of tau was differentially affected. In GD conditions, tau was found dephosphorylated at Ser199, Thr205, and Ser404 and hyperphosphorylated at Ser262. However, in OD conditions tau was found hyperphosphorylated at Thr205, SerSer356, Ser396, and Ser404. Combined OD and GD resulted in degradation of cellular tau and dephosphorylation of PSD tau at Ser396 and Ser404. These results indicate that oxygen deprivation causes dephosphorylation of tau, while GD and OD differentially affect distribution of total tau and tau phosphorylation variants in neuronal compartments by activating different mechanisms.

An integrated bacterial system for the discovery of chemical rescuers of disease-associated protein misfolding.

Protein misfolding and aggregation are common pathological features of several human diseases, including Alzheimer's disease and type 2 diabetes. Here, we report an integrated and generalizable bacterial system for the facile discovery of chemical rescuers of disease-associated protein misfolding. In this system, large combinatorial libraries of macrocyclic molecules are biosynthesized in Escherichia coli cells and simultaneously screened for their ability to rescue pathogenic protein misfolding and aggregation using a flow cytometric assay. We demonstrate the effectiveness of this approach by identifying drug-like, head-to-tail cyclic peptides that modulate the aggregation of the Alzheimer's disease-associated amyloid ß peptide. Biochemical, biophysical and biological assays using isolated amyloid ß peptide, primary neurons and various established Alzheimer's disease nematode models showed that the selected macrocycles potently inhibit the formation of neurotoxic amyloid ß peptide aggregates. We also applied the system to the identification of misfolding rescuers of mutant Cu/Zn superoxide dismutase-an enzyme linked with inherited forms of amyotrophic lateral sclerosis. Overall, the system enables the identification of molecules with therapeutic potential for rescuing the misfolding of disease-associated polypeptides.

Synthesis and evaluation of gallocyanine dyes as potential agents for the treatment of Alzheimer's disease and related neurodegenerative tauopathies.

In search of safe and effective anti-Alzheimer disease agents a series of gallocyanine dyes have been synthesized and evaluated for their ability to inhibit LRPs/DKK1 interactions. Modulation of the interactions between LRPS and DKK1, regulate Wnt signaling pathway and affect Tau phosphorylation. The current efforts resulted in the identification of potent DKK1 inhibitors which are able to inhibit prostaglandin J2-induced tau phosphorylation at serine 396.

Structural and regulatory elements of the interaction between amyloid-ß protein precursor and Homer3.

Amyloid-ß protein precursor (AßPP) metabolism and the accumulation of its derivative amyloid-ß (Aß) peptide in senile plaques have been considered key players in the development of Alzheimer's disease (AD). However, the mechanisms underlying the generation and the deposition of Aß are not clear but emphasis has been given in the role of AßPP protein interactions that regulate its processing and offer a means to manipulate Aß production. We have previously shown that AßPP interacts with members of the Homer protein family, which leads to inhibition of Aß generation. Herein, we studied the structural parameters of AßPP/Homer3 interaction by analyzing the sequences and domains that play a role in the formation of the complex. We found that the cytoplasmic tail of AßPP is necessary for the interaction. Regarding Homer3, we report that both the EVH1 protein interacting domain and the polymerization coiled coil domain are essential for the complex assembly. Importantly, phosphorylation of Homers at certain serine residues seems to enhance the interaction with AßPP, possibly underlying our recent work suggesting that calcium signaling also regulates the interaction. Our results show that the regulation of AßPP/Homer3 interaction might be critical in the context of Alzheimer's disease pathology as a novel target for regulating AßPP function and metabolism.

Calcium regulates the interaction of amyloid precursor protein with Homer3 protein.

Ca(2+) dysregulation is an important factor implicated in Alzheimer's disease pathogenesis. The mechanisms mediating the reciprocal regulation of Ca(2+) homeostasis and amyloid precursor protein (APP) metabolism, function, and protein interactions are not well known. We have previously shown that APP interacts with Homer proteins, which inhibit APP processing toward amyloid-ß. In this study, we investigated the effect of Ca(2+) homeostasis alterations on APP/Homer3 interaction. Influx of extracellular Ca(2+) upon treatment of HEK293 cells with the ionophore A23187 or addition of extracellular Ca(2+) in cells starved of calcium specifically reduced APP/Homer3 but not APP/X11a interaction. Endoplasmic reticulum Ca(2+) store depletion by thapsigargin followed by store-operated calcium entry also decreased the interaction. Interestingly, application of a phospholipase C stimulator, which causes inositol 1,4,5-trisphosphate-induced endoplasmic reticulum Ca(2+) release, caused dissociation of APP/Homer3 complex. In human neuroblastoma cells, membrane depolarization also disrupted the interaction. This is the first study showing that changes in Ca(2+) homeostasis affect APP protein interactions. Our results suggest that Ca(2+) and Homers play a significant role in the development of Alzheimer's disease pathology.

BRI2 interacts with BACE1 and regulates its cellular levels by promoting its degradation and reducing its mRNA levels.

BRI2, a protein mutated in Familial British and Familial Danish Dementias, interacts with Amyloid Precursor Protein (APP) and reduces the levels of secreted APPß (sAPPß), which derives from APP cleavage by ß-secretase (BACE1). Exploring the mechanisms of this effect, we obtained data that BRI2 decreases the cellular levels of BACE1 thus reducing the ß-cleavage of APP. Deletion of N-terminal cytoplasmic or C-terminal extracellular sequences of BRI2 neither affected its interaction with BACE1 or APP (Fotinopoulou et al., 2005) nor the reduction in the levels of BACE1 and sAPPß. These results suggest that BRI2 may prevent access of BACE1 to APP and the BRI2/BACE1 interaction may mediate the reduction in BACE1 levels. In support, BRI2 expression induced lysosomal but not proteasomal degradation of BACE1. In parallel, BRI2 expression was also found to reduce BACE1 mRNA levels by 50%. This study adds novel information regarding the mechanism by which BRI2 affects APP processing and BACE1 levels.

Downregulation of AßPP enhances both calcium content of endoplasmic reticulum and acidic stores and the dynamics of store operated calcium channel activity.

The amyloid-ß protein precursor (AßPP) is a type-1 transmembrane protein involved in Alzheimer's disease (AD). It has become increasingly evident that AßPP, its protein-protein interactions, and its proteolytical fragments may affect calcium homeostasis and vice versa. In addition, there is evidence that calcium dysregulation contributes to AD. To study the role of AßPP in calcium homeostasis, we downregulated its expression in SH-SY5Y cells using shRNA (SH-SY5Y/AßPP-) or increased expression of AßPP695 by transfection (SH-SY5Y/AßPP+). The levels of cytosolic Ca2+ after treatment with thapsigargin, monensin, activation of capacitative calcium entry (CCE), and treatment with SKF, a store operated channel (SOCs) inhibitor, were measured by fura-2AM fluorimetry. SH-SY5Y/AßPP+ cells show reduced response to thapsigargin and reduced CCE, although this reduction is not statistically significant. On the other hand, we found that, relative to SH-SY5Y, SH-SY5Y/AßPP- cells show a significant increase in the response to thapsigargin but not in CCE and their SOCs were more susceptible to SKF inhibition. Additionally, downregulation of AßPP resulted in increased response to monensin that induces calcium release from acidic stores. The increase of calcium release from the endoplasmic reticulum and the acidic stores, when AßPP is downregulated, could be attributed to elevated Ca2+ content or to a dysregulation of Ca2+ transfer through their membranes. These data, along with already existing evidence regarding the role of AßPP in calcium homeostasis and the early occurring structural and functional abnormalities of endosomes, further substantiate the role of AßPP in calcium homeostasis and in AD.

Cell-line specific protection by berry polyphenols against hydrogen peroxide challenge and lack of effect on metabolism of amyloid precursor protein.

Amyloid precursor protein (APP) altered metabolism, Aß-overproduction/aggregation and oxidative stress are implicated in the development of Alzheimer's disease pathology. Based on our previous data indicating that administration of a polyphenol-rich (PrB) blueberry extract (from wild Vaccinium angustifolium) is memory enhancing in healthy mice and in order to delineate the neuroprotective mechanisms, this study investigated the antioxidant effects of PrB in H2O2-induced oxidative damage, Aß peptide fibrillogenesis and APP metabolism. PrB suppressed H2O2-initiated oxidation (DCF assay) and cell death (MTT assay) in SH-SY5Y cells. Protective effects were observed on Chinese hamster ovary (CHO) cells overexpressing APP770 carrying the mutation Val717Phe only at high concentrations, while further damage on HEK293 cells was induced after co-treatment with 250 µM H2O2 and PrB in comparison with H2O2 alone. Using the thioflavine T assay, blueberry polyphenols inhibited Aß-aggregation (~70%, 15 µg/mL) in a time-dependent manner, while in the CHO(APP770) cells it had no effect on APP metabolism as assessed by western blot. The results suggest that blueberry polyphenols exhibit antioxidant and/or pro-oxidant properties according to the cellular environment and have no effect on APP metabolism.

Glycosylation of BRI2 on asparagine 170 is involved in its trafficking to the cell surface but not in its processing by furin or ADAM10.

Two different mutated forms of BRI2 protein are linked with familial British and Danish dementias, which present neuropathological similarities with Alzheimer's disease. BRI2 is a type II transmembrane protein that is trafficked through the secretory pathway to the cell surface and is processed by furin and ADAM10 (a disintegrin and metalloproteinase domain 10) to release secreted fragments of unknown function. Its apparent molecular mass (42-44 kDa) is significantly higher than that predicted by the number and composition of amino acids (30 kDa) suggesting that BRI2 is glycosylated. In support, bioinformatics analysis indicated that BRI2 bears the consensus sequence Asn-Thr-Ser (residues 170-173) and could be N-glycosylated at Asn170. Given that N-glycosylation is considered essential for protein folding, processing and trafficking, we examined whether BRI2 is N-glycosylated. Treatment of HEK293 (human embryonic kidney) cells expressing BRI2 with the N-glycosylation inhibitor tunicamycin or mutation of Asn170 to alanine reduced its molecular mass by ~2 kDa. These data indicate that BRI2 is N-glycosylated at Asn170. To examine the effect of N-glycosylation on BRI2 trafficking at the cell surface, we performed biotinylation and (35)S methionine pulse-chase experiments. These experiments showed that mutation of Asn170 to alanine reduced BRI2 trafficking at the cell surface and its steady state levels at the plasma membrane. Furthermore, we obtained data indicating that this mutation did not affect cleavage of BRI2 by furin or ADAM10. Our results confirm the theoretical predictions that BRI2 is N-glycosylated at Asn170 and show that this post-translational modification is essential for its expression at the cell surface but not for its proteolytic processing.

Memory enhancing effects of saffron in aged mice are correlated with antioxidant protection.

Brain aging is characterized by cognitive decline and memory deficits that could be the result of oxidative stress and impaired cholinergic function. In this study, the effects of a daily, 7-day, intraperitoneal administration of saffron on cognitive functions were examined in both healthy adult (4 months old) and aged (20 months old), male Balb-c mice (n=8/group), by passive avoidance test. Whole brain homogenates (minus cerebellum) were collected for examination of brain oxidative markers, caspase-3 and acetylcholinesterase (AChE) activity. Results showed that saffron-treated mice exhibited significant improvement in learning and memory, accompanied by reduced lipid peroxidation products, higher total brain antioxidant activity and reduced caspase-3 activity in both age groups of mice. Furthermore, salt- and detergent-soluble AChE activity was significantly decreased only in adult mice. Thus, we showed, for the first time, that the significant cognitive enhancement conferred by saffron administration in mice, is more closely related to the antioxidant reinforcement. Next, we compared the effect of saffron (1-250 µg/mL), crocetin and safranal (1-125 µM) on H(2)O(2)-induced toxicity in human neuroblastoma SH-SY5Y cells. Both saffron and crocetin provided strong protection in rescuing cell viability (MTT assay), repressing ROS production (DCF assay) and decreasing caspase-3 activation. These data, together with earlier studies suggest that crocetin is a unique and potent antioxidant, capable of mediating the in vivo effects of saffron.

BRI2 homodimerizes with the involvement of intermolecular disulfide bonds.

Familial British and Familial Danish Dementia (FBD and FDD) are two dominantly inherited neurodegenerative diseases that present striking similarities with Alzheimer's disease. The genetic defects underlying those dementias are mutations in the gene that encodes for BRI2 protein. Cleavage of mutated BRI2 by furin releases the peptides ABri or ADan, which accumulate in the brains of patients. BRI2 normal function is yet unknown. To unwind aspects of its cellular role, we investigated the possibility that BRI2 forms dimers, based on structural elements of the protein, the GXXXG motif within its transmembrane domain and the odd number of cysteine residues. We found that BRI2 dimerizes in cells and that dimers are held via non-covalent interactions and via disulfide bridges between the cysteines at position 89. Additionally, we showed that BRI2 dimers are formed in the ER and appear at the cell surface. Finally, BRI2 dimers were found to exist in mouse brain. Revealing the physiological properties of BRI2 is critical in the elucidation of the deviations that lead to neurodegeneration.

BRI2 as a central protein involved in neurodegeneration.

BRI2 is a protein that when mutated causes familial British and familial Danish dementias. Upon cleavage, the mutated BRI2 proteins release the peptides ABri and ADan, which are amyloidogenic and accumulate in the brains of patients. Although BRI2 has an unknown function, several reports indicate that it could play multiple roles. For example, the fact that it exists at the cell surface as a homodimer indicates that it could be involved in cell signaling events by acting as a receptor. BRI2 also interacts with amyloid precursor protein (APP), involved in Alzheimer's disease (AD). In cell cultures and mouse models of AD, BRI2 inhibits APP processing and reduces amyloid beta peptide deposition. The interaction between the two proteins could be responsible for the neuropathological similarities between familial British/Danish dementias and AD. The study of BRI2, which is central in familial British and Danish dementia, could unravel underlying molecular mechanisms of neurodegeneration.

Homer2 and Homer3 interact with amyloid precursor protein and inhibit Abeta production.

The study of Amyloid Precursor Protein (APP) processing has been the focus of considerable interest, since it leads to Abeta peptide generation, the main constituent of neuritic plaques found in brains of Alzheimer's disease patients. Therefore, the identification of novel APP binding partners that regulate Abeta peptide production represents a pharmaceutical target aiming at reducing Alphabeta pathology. In this study, we provide evidence that Homer2 and Homer3 but not Homer1 proteins interact specifically with APP. Their expression inhibits APP processing and reduces secretion of Abeta peptides. In addition, they decrease the levels of cell surface APP and inhibit maturation of APP and beta-secretase (BACE1). The effects of Homer2 and Homer3 on APP trafficking to the cell surface and/or on APP and BACE1 maturation could be part of the mechanism by which the expression of these proteins leads to the significant reduction of Abeta peptide production.

Abeta(1-40)-induced secretion of matrix metalloproteinase-9 results in sAPPalpha release by association with cell surface APP.

To understand matrix metalloproteinase-9 (MMP-9) involvement in Alzheimer's disease, we examined mechanisms mediating increased expression of MMP-9 in the presence of Abeta(1-40) and the role of MMP-9 on amyloid precursor protein (APP) processing. Up-regulation of MMP-9 expressed by SK-N-SH cells in the presence of Abeta(1-40) was mediated by alpha(3)beta(1) and alpha(2)beta(1) integrin receptors. Overexpression of MMP-9 or treatment of HEK/APP695 cells with activated recombinant MMP-9 resulted in enhanced secretion of soluble APP (sAPPalpha), a product of alpha-secretase cleavage, and reduction of Abeta release. MMP-9 effect was enhanced by phorbol 12-mysistrate-13-acetate (PMA), an alpha-secretase activator and inhibited by EDTA or SB-3CT, an MMP-9 inhibitor. Additionally, immunoprecipitation and confocal microscopy demonstrated that MMP-9 and APP695 were associated on the cell surface. These results indicate that Abeta peptide increases MMP-9 secretion through integrins; MMP-9 then directly processes cell surface APP695 with an alpha-secretase like activity, substantially reducing the levels of secreted Abeta peptide.

Novel processing of Notch 1 within its intracellular domain by a cysteine protease.

In order to study N1 processing, we expressed human N1 (hN1) in HEK293 cells (293-hN1). Following Western blot analysis of 293-hN1 extracts, we detected, in addition to full-length hN1 and the N1 extracellular domain truncated form (N1-TM), a novel extracellular domain truncated form of hN1 with a COOH-terminal deletion, designated hN1-TMdeltaCT. Treatment of cells with the gamma-secretase inhibitor L-685,458 resulted in an accumulation of hN1-TMdeltaCT suggesting that this fragment is a gamma-secretase substrate. To identify the proteolytic activity(ies) that generates hN1-TMdeltaCT, we treated 293-hN1 cells with inhibitors of proteasome, calpains, caspases, serine and cysteine proteases. Despite the presence of a caspase-3 cleavage site within hN1 intracellular domain, none of the caspase inhibitors inhibited hN1-TMdeltaCT production. The proteasomal inhibitors used had also no effect. Incubation of cells with the cysteine protease inhibitor E64d resulted in the accumulation of hN1-TM and the inhibition of hN1-TMdeltaCT production suggesting a precursor-product relationship and that a cysteine protease is involved. Similarly, treatment of cells expressing amyloid precursor protein or E-cadherin with E-64d resulted in the accumulation of COOH-terminal fragments suggesting that these proteins are also processed within their intracellular domain by a cysteine protease. Processing towards hN1-TMdeltaCT requires maturation and transport of hN1 to the cell surface since treatment with brefeldin A inhibited its production and resulted in accumulation of hN1. Processing of hN1 within its intracellular domain could generate fragments that can exert novel functions and/or interfere with the function of hN1 intracellular domain.