Center of Biomedical Research Excellence in Matrix Biology: Building Research Infrastructure, Supporting Young Researchers, and Fostering Collaboration.

The Center of Biomedical Research Excellence in Matrix Biology strives to improve our understanding of extracellular matrix at molecular, cellular, tissue, and organismal levels to generate new knowledge about pathophysiology, normal development, and regenerative medicine. The primary goals of the Center are to i) support junior investigators, ii) enhance the productivity of established scientists, iii) facilitate collaboration between both junior and established researchers, and iv) build biomedical research infrastructure that will support research relevant to cell-matrix interactions in disease progression, tissue repair and regeneration, and v) provide access to instrumentation and technical support. A Pilot Project program provides funding to investigators who propose applying their expertise to matrix biology questions. Support from the National Institute of General Medical Sciences at the National Institutes of Health that established the Center of Biomedical Research Excellence in Matrix Biology has significantly enhanced the infrastructure and the capabilities of researchers at Boise State University, leading to new approaches that address disease diagnosis, prevention, and treatment. New multidisciplinary collaborations have been formed with investigators who may not have previously considered how their biomedical research programs addressed fundamental and applied questions involving the extracellular matrix. Collaborations with the broader matrix biology community are encouraged.

Intranasal delivery of shRNA to knockdown the 5HT-2A receptor enhances memory and alleviates anxiety.

Short-hairpin RNAs (shRNA), targeting knockdown of specific genes, hold enormous promise for precision-based therapeutics to treat numerous neurodegenerative disorders. However, whether shRNA constructed molecules can modify neuronal circuits underlying certain behaviors has not been explored. We designed shRNA to knockdown the human HTR2A gene in vitro using iPSC-differentiated neurons. Multi-electrode array (MEA) results showed that the knockdown of the 5HT-2A mRNA and receptor protein led to a decrease in spontaneous electrical activity. In vivo, intranasal delivery of AAV9 vectors containing shRNA resulted in a decrease in anxiety-like behavior in mice and a significant improvement in memory in both mice (104%) and rats (92%) compared to vehicle-treated animals. Our demonstration of a non-invasive shRNA delivery platform that can bypass the blood-brain barrier has broad implications for treating numerous neurological mental disorders. Specifically, targeting the HTR2A gene presents a novel therapeutic approach for treating chronic anxiety and age-related cognitive decline.

Proteolytic cleavage of apolipoprotein E4 as the keystone for the heightened risk associated with Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by microscopic lesions consisting of beta-amyloid plaques and neurofibrillary tangles (NFTs). The majority of cases are defined as sporadic and are likely caused by a combination of both genetic and environmental factors. Of the genetic risk factors identified, the 34 kDa protein, apolipoprotein (apo) E4, is of significant importance as APOE4 carriers account for 65%-80% of all AD cases. Although apoE4 plays a normal role in lipoprotein transport, how it contributes to AD pathogenesis is currently unknown. One potential mechanism by which apoE4 contributes to disease risk is its propensity to undergo proteolytic cleavage generating N- and C-terminal fragments. The purpose of this review will be to examine the mechanisms by which apoE4 contributes to AD pathogenesis focusing on the potential loss or gain of function that may occur following cleavage of the full-length protein. In this context, a discussion of whether targeting apoE4 therapeutically is a rationale approach to treating this disease will be assessed.

Genetic modulation of the HTR2A gene reduces anxiety-related behavior in mice.

The expanding field of precision gene editing using CRISPR/Cas9 has demonstrated its potential as a transformative technology in the treatment of various diseases. However, whether this genome-editing tool could be used to modify neural circuits in the central nervous system (CNS), which are implicated in complex behavioral traits, remains uncertain. In this study, we demonstrate the feasibility of noninvasive, intranasal delivery of adeno-associated virus serotype 9 (AAV9) vectors containing CRISPR/Cas9 cargo within the CNS resulting in modification of the HTR2A receptor gene. In vitro, exposure to primary mouse cortical neurons to AAV9 vectors targeting the HT2RA gene led to a concentration-dependent decrease in spontaneous electrical activity following multielectrode array (MEA) analysis. In vivo, at 5 weeks postintranasal delivery in mice, analysis of brain samples revealed single base pair deletions and nonsense mutations, leading to an 8.46-fold reduction in mRNA expression and a corresponding 68% decrease in the 5HT-2A receptor staining. Our findings also demonstrate a significant decrease in anxiety-like behavior in treated mice. This study constitutes the first successful demonstration of a noninvasive CRISPR/Cas9 delivery platform, capable of bypassing the blood-brain barrier and enabling modulation of neuronal 5HT-2A receptor pathways. The results of this study targeting the HTR2A gene provide a foundation for the development of innovative therapeutic strategies for a broad range of neurological disorders, including anxiety, depression, attentional deficits, and cognitive dysfunction.

An amino-terminal fragment of apolipoprotein E4 leads to behavioral deficits, increased PHF-1 immunoreactivity, and mortality in zebrafish.

Although the increased risk of developing sporadic Alzheimer's disease (AD) associated with the inheritance of the apolipoprotein E4 (APOE4) allele is well characterized, the molecular underpinnings of how ApoE4 imparts risk remains unknown. Enhanced proteolysis of the ApoE4 protein with a toxic-gain of function has been suggested and a 17 kDa amino-terminal ApoE4 fragment (nApoE41-151) has been identified in post-mortem human AD frontal cortex sections. Recently, we demonstrated in vitro, exogenous treatment of nApoE41-151 in BV2 microglial cells leads to uptake, trafficking to the nucleus and increased expression of genes associated with cell toxicity and inflammation. In the present study, we extend these findings to zebrafish (Danio rerio), an in vivo model system to assess the toxicity of nApoE41-151. Exogenous treatment of nApoE41-151 to 24-hour post-fertilization for 24 hours resulted in significant mortality. In addition, developmental abnormalities were observed following treatment with nApoE41-151 including improper folding of the hindbrain, delay in ear development, deformed yolk sac, enlarged cardiac cavity, and significantly lower heart rates. A similar nApoE31-151 fragment that differs by a single amino acid change (C>R) at position 112 had no effects on these parameters under identical treatment conditions. Decreased presence of pigmentation was noted for both nApoE31-151- and nApoE41-151-treated larvae compared with controls. Behaviorally, touch-evoked responses to stimulus were negatively impacted by treatment with nApoE41-151 but did not reach statistical significance. Additionally, triple-labeling confocal microscopy not only confirmed the nuclear localization of the nApoE41-151 fragment within neuronal populations following exogenous treatment, but also identified the presence of tau pathology, one of the hallmark features of AD. Collectively, these in vivo data demonstrating toxicity as well as sublethal effects on organ and tissue development support a novel pathophysiological function of this AD associated-risk factor.

Depletion of Beclin-1 due to proteolytic cleavage by caspases in the Alzheimer's disease brain.

The Beclin-1 protein is essential for the initiation of autophagy, and recent studies suggest this function may be compromised in Alzheimer's disease (AD). In addition, in vitro studies have supported a loss of function of Beclin-1 due to proteolytic modification by caspases. In the present study, we examined whether caspase-cleavage of Beclin-1 occurs in the AD brain by designing a site-directed caspase-cleavage antibody based upon a known cleavage site within the protein at position D149. We confirmed that Beclin-1 is an excellent substrate for caspase-3 and demonstrates cleavage led to the formation of a 35-kDa C-terminal fragment labeled by our novel antibody following Western blot analysis. Application of this antibody termed Beclin-1 caspase-cleavage product antibody or BeclinCCP in frontal cortex tissue sections revealed strong immunolabeling within astrocytes that localized with plaque regions and along blood vessels in all AD cases examined. In addition, weaker, more variable BeclinCCP labeling was also observed within neurofibrillary tangles that colocalized with the early tau conformational marker, MC-1 as well as the late tangle marker, PHF-1. Collectively, these data support a depletion of Beclin-1 in AD following caspase-cleavage. This article is part of a Special Issue entitled "Autophagy and protein degradation in neurological diseases."

Fragmentation of Apolipoprotein E4 is Required for Differential Expression of Inflammation and Activation Related Genes in Microglia Cells.

The apolipoprotein E4 (APOE4) allele represents the single greatest risk factor for late-onset Alzheimer's disease (AD) and accumulating evidence suggests that fragmentation with a toxic-gain of function may be a key molecular step associated with this risk. Recently, we demonstrated strong immunoreactivity of a 151 amino-terminal fragment of apoE4 (E4-fragment) within the nucleus of microglia in the human AD brain. In vitro, this fragment led to toxicity and activation of inflammatory processes in BV2 microglia cells. Additionally, a transcriptome analysis following exogenous treatment of BV2 microglia cells with this E4 fragment led to a > 2-fold up regulation of 1,608 genes, with many genes playing a role in inflammation and microglia activation. To extend these findings, we here report a similar transcriptome analysis in BV2 microglia cells following treatment with full-length ApoE4 (FL-ApoE4). The results indicated that full-length ApoE4 had a very small effect on gene expression compared to the fragment. Only 48 differentially expressed genes (DEGs) were identified (p < 0.05, and greater than 2-fold change). A gene ontology analysis of these DEGs indicated that they are not involved in inflammatory and activation processes, in contrast to the genes up regulated by the E4-fragment. In addition, genes that showed a negative fold-change upon FL-E4 treatment typically showed a strong positive fold-change upon treatment with the fragment (Pearson's r = -0.7). Taken together, these results support the hypothesis that a key step in the conversion of microglia to an activated phenotype is proteolytic cleavage of FL-ApoE4. Therefore, the neutralization of this amino-terminal fragment of ApoE4, specifically, may serve as an important therapeutic strategy in the treatment of AD.

The role of caspases in Alzheimer's disease; potential novel therapeutic opportunities.

Although apoptosis plays a critical role in molding the CNS into its final appearance and function, inappropriate activation of this pathway in the aging brain may contribute to neurodegeneration. In Alzheimer's disease (AD), an overwhelming body of evidence supports the activation of apoptosis in general, and caspases specifically as an early event that may not only contribute to neurodegeneration but also promote the underlying pathology associated with this disease. Therefore, caspase inhibitors may provide an effective strategy for treating AD. However, despite the compelling evidence indicating a role for caspases in disease progression, chronic treatment with caspase inhibitors in animal models of AD has never been undertaken. In this review the role of caspases in AD will be addressed, including recent studies utilizing in vivo transgenic mouse models of tauopathies. In addition, a discussion of the therapeutic value and dangers of targeting caspase inhibition in the treatment of AD using caspase inhibitors such as Q-VD-OPh will be evaluated.

Caspase-cleaved transactivation response DNA-binding protein 43 in Parkinson's disease and dementia with Lewy bodies.

BACKGROUND: Transactivation response DNA-binding protein 43 (TDP-43) proteinopathies are classified based upon the extent of modified TDP-43 and include a growing number of neurodegenerative diseases such as amyotrophic lateral sclerosis, frontotemporal lobar degeneration with ubiquitin-immunoreactive, tau-negative inclusions and frontotemporal lobar degeneration with motor neuron disease. OBJECTIVE: The purpose of the study was to examine whether proteolytic modifications of TDP-43 are a relevant finding in Parkinson's disease (PD) and dementia with Lewy bodies (DLB). METHODS: A novel site-directed caspase cleavage antibody, termed TDP caspase cleavage product antibody (TDPccp), was utilized based upon a known caspase 3 cleavage consensus site within TDP-43 at position 219. RESULTS: Application of this antibody to postmortem brain sections from PD and DLB patients revealed the presence of caspase-cleaved TDP-43 in Lewy bodies and Hirano bodies in all cases examined. Colocalization of TDPccp with an antibody to alpha-synuclein (alpha-Syn), which served as a general marker for Lewy bodies, was evident within the substantia nigra in both alpha-synucleinopathies. Interestingly, the TDPccp antibody detected a greater number of Lewy bodies in PD and DLB compared to the alpha-Syn antibody. In addition, a semiquantitative analysis in both diseases confirmed this finding by indicating that the percentage of caspase-cleaved TDP-43 single-labeled Lewy bodies was approximately twice that of alpha-Syn labeling (in DLB 13.4 vs. 5.5%, while in PD 34.6 vs. 17.6%). CONCLUSION: Collectively, these data have identified caspase-cleaved TDP-43 as a primary component of Lewy and Hirano bodies in PD and DLB, and suggest that the TDPccp antibody is an effective marker for the detection of Lewy bodies in these neurodegenerative diseases.

Clinical Trials in Alzheimer's Disease: A Hurdle in the Path of Remedy.

Human clinical trials seek to ameliorate the disease states and symptomatic progression of illnesses that, as of yet, are largely untreatable according to clinical standards. Ideally, clinical trials test "disease-modifying drugs," i.e., therapeutic agents that specifically modify pathological features or molecular bases of the disease and would presumably have a large impact on disease progression. In the case of Alzheimer's disease (AD), however, this approach appears to have stalled progress in the successful development of clinically useful therapies. For the last 25 years, clinical trials involving AD have centered on beta-amyloid (Aß) and the Aß hypothesis of AD progression and pathology. According to this hypothesis, the progression of AD begins following an accumulation of Aß peptide, leading to eventual synapse loss and neuronal cell death: the true overriding pathological feature of AD. Clinical trials arising from the Aß hypothesis target causal steps in the pathway in order to reduce the formation of Aß or enhance clearance, and though agents have been successful in this aim, they remain unsuccessful in rescuing cognitive function or slowing cognitive decline. As such, further use of resources in the development of treatment options for AD that target Aß, its precursors, or its products should be reevaluated. The purpose of this review was to give an overview of how human clinical trials are conducted in the USA and to assess the results of recent failed trials involving AD, the majority of which were based on the Aß hypothesis. Based on these current findings, it is suggested that lowering Aß is an unproven strategy, and it may be time to refocus on other targets for the treatment of this disease including pathological forms of tau.

Transcriptome Analyses in BV2 Microglial Cells Following Treatment With Amino-Terminal Fragments of Apolipoprotein E.

Despite the fact that harboring the apolipoprotein E4 (APOE4) allele represents the single greatest risk factor for late-onset Alzheimer's disease (AD), the exact mechanism by which ApoE4 contributes to disease progression remains unknown. Recently, we demonstrated that a 151 amino-terminal fragment of ApoE4 (nApoE41-151) localizes within the nucleus of microglia in the human AD brain and traffics to the nucleus causing toxicity in BV2 microglia cells. In the present study, we examined in detail what genes may be affected following treatment by nApoE41-151. Transcriptome analyses in BV2 microglial cells following sublethal treatment with nApoE41-151 revealed the upregulation of almost 4,000 genes, with 20 of these genes upregulated 182- to 715-fold compared to untreated control cells. The majority of these 20 genes play a role in the immune response and polarization toward microglial M1 activation. As a control, an identical nApoE31-151 fragment that differed by a single amino acid at position 112 (Cys→Arg) was tested and produced a similar albeit lower level of upregulation of an identical set of genes. In this manner, enriched pathways upregulated by nApoE31-151 and nApoE41-151 following exogenous treatment included Toll receptor signaling, chemokine/cytokine signaling and apoptosis signaling. There were unique genes differentially expressed by at least two-fold for either fragment. For nApoE31-151, these included 16 times as many genes, many of which are involved in physiological functions within microglia. For nApoE41-151, on the other hand the number genes uniquely upregulated was significantly lower, with many of the top upregulated genes having unknown functions. Taken together, our results suggest that while nApoE31-151 may serve a more physiological role in microglia, nApoE41-151 may activate genes that contribute to disease inflammation associated with AD. These data support the hypothesis that the link between harboring the APOE4 allele and dementia risk could be enhanced inflammation through activation of microglia.

Lack of pathology in a triple transgenic mouse model of Alzheimer's disease after overexpression of the anti-apoptotic protein Bcl-2.

Alzheimer's disease (AD) is characterized by the accumulation of plaques containing beta-amyloid (Abeta) and neurofibrillary tangles (NFTs) consisting of modified tau. Although Abeta deposition is thought to precede the formation of NFTs in AD, the molecular steps connecting these two pathologies is not known. Previous studies have suggested that caspase activation plays an important role in promoting the pathology associated with AD. To further understand the contribution of caspases in disease progression, a triple transgenic Alzheimer's mouse model overexpressing the anti-apoptotic protein Bcl-2 was generated. Here we show that overexpression of Bcl-2 limited caspase-9 activation and reduced the caspase cleavage of tau. Moreover, overexpression of Bcl-2 attenuated the processing of APP (amyloid precursor protein) and tau and reduced the number of NFTs and extracellular deposits of Abeta associated with these animals. In addition, overexpression of Bcl-2 in 3xTg-AD mice improved place recognition memory. These findings suggest that the activation of apoptotic pathways may be an early event in AD and contributes to the pathological processes that promote the disease mechanisms underlying AD.

A Fragment of Apolipoprotein E4 Leads to the Downregulation of a CXorf56 Homologue, a Novel ER-Associated Protein, and Activation of BV2 Microglial Cells.

Despite the fact that harboring the apolipoprotein E4 (APOE4) allele represents the single greatest risk factor for late-onset Alzheimer's disease (AD), the exact mechanism by which apoE4 contributes to disease progression remains unknown. Recently, we demonstrated that a 151 amino-terminal fragment of apoE4 (nApoE41-151) localizes within the nucleus of microglia in the human AD brain, suggesting a potential role in gene expression. In the present study, we investigated this possibility utilizing BV2 microglia cells treated exogenously with nApoE41-151. The results indicated that nApoE41-151 leads to morphological activation of microglia cells through, at least in part, the downregulation of a novel ER-associated protein, CXorf56. Moreover, treatment of BV2 cells with nApoE41-151 resulted in a 68-fold increase in the expression of the inflammatory cytokine, TNFα, a key trigger of microglia activation. In this regard, we also observed a specific binding interaction of nApoE41-151 with the TNFα promoter region. Collectively, these data identify a novel gene-regulatory pathway involving CXorf56 that may link apoE4 to microglia activation and inflammation associated with AD.

Caspase activation in Alzheimer's disease: early to rise and late to bed.

It has been almost 15 years since the first report of apoptosis as a major mechanism of cell death associated with Alzheimer's disease (AD). Presently, whether neurons die through apoptosis or some other pathway is still a hotly debated issue. However, mounting evidence suggests a role for the activation of caspases and cleavage of critical cellular proteins during the progression of AD. The activation of apoptotic pathways may represent a protracted battle due to the presence of various anti-apoptotic molecules such as Bcl-2 whereby neurons do not immediately execute the apoptotic program but caspase activation occurs discretely at some low level. During this time, caspase cleavage of the amyloid precursor protein (APP), the adaptor protein GGA3 involved with beta-amyloid production, and tau may promote the formation of beta-amyloid (A beta) and neurofibrillary tangles (NFTs). Thus, not only may activation of caspases represent a terminal event associated with AD (i.e. cell death), but also a proximal event that promotes the pathology underlying this disease. Therefore, therapeutics aimed at preventing the activation and execution of apoptosis may provide an effective means of treating AD.

Caspase-cleaved TAR DNA-binding protein-43 is a major pathological finding in Alzheimer's disease.

The TAR DNA-binding protein-43 (TDP-43) has been identified as a major constituent of inclusions found in frontotemporal dementia with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). To determine a possible role for TDP-43 in Alzheimer's disease (AD), a site-directed caspase-cleavage antibody to TDP-43 based upon a known caspase-3 cleavage consensus site within TDP- 43 at position D219 was designed. In vitro, this antibody labeled the predicted 25 kDa caspase-cleavage fragment of TDP-43 without labeling full-length TDP-43 following digestion of recombinant TDP-43 with caspase-3 or treatment of HeLa cells with staurosporine. Application of this antibody in postmortem brain sections indicated the presence of caspase-cleaved TDP-43 in Hirano bodies, tangles, reactive astrocytes and neuritic plaques of the AD brain. Caspase-cleaved TDP-43 also co-localized with ubiquitin labeled neurons as well as dystrophic neurites within plaque regions. These results suggest that caspase-cleaved TDP-43 is a major pathological finding in AD and may contribute to the neurodegeneration associated with this disease.

Apolipoprotein E Fragmentation within Lewy Bodies of the Human Parkinson's Disease Brain.

Although harboring the Apolipoprotein E4 (APOE4) allele is a well-known risk factor in Alzheimer's disease (AD), whether a similar risk holds true for Parkinson's disease (PD) is currently not known. To investigate whether apoE pathology is present in PD, an immunohistochemical study was undertaken with fixed, human PD brain sections from the substantia nigra utilizing a recently characterized antibody that detects an amino-terminal fragment of apoE. This antibody, termed the apoE cleavage fragment p17 (nApoECFp17) antibody specifically detects an amino-terminal 17 kDa fragment of apoE without reacting with full-length forms of the protein. Application of this antibody revealed the presence of this fragment in Lewy bodies in all cases examined. Colocalization of nApoECFp17 with an antibody to alpha-synuclein (α-Syn), which served as a general marker for Lewy bodies, indicated the presence of this apoE fragment in 87.5% of all identified Lewy bodies. In addition, localization of nApoECFp17 was also evident within oligodendrocytes, the nucleus of melatonin-containing neurons, and blood vessels. Conversely, little staining was observed in the substantia nigra from Pick's disease or in the frontal cortex of dementia with Lewy bodies (DLB) cases, suggesting a specificity for nApoECFp17 immunoreactivity in PD. Collectively, these data have identified widespread evidence for apoE fragmentation in the human PD brain and documented for the first time the presence of apoE within Lewy bodies, the major pathological marker for this neurodegenerative disease.

The Potential of CRISPR/Cas9 Gene Editing as a Treatment Strategy for Alzheimer's Disease.

Despite a wealth of knowledge gained in the past three decades concerning the molecular underpinnings of Alzheimer's disease (AD), progress towards obtaining effective, disease modifying therapies has proven to be challenging. In this manner, numerous clinical trials targeting the production, aggregation, and toxicity of beta-amyloid, have failed to meet efficacy standards. This puts into question the beta-amyloid hypothesis and suggests that additional treatment strategies should be explored. The recent emergence of CRISPR/Cas9 gene editing as a relatively straightforward, inexpensive, and precise system has led to an increased interest of applying this technique in AD. CRISPR/Cas9 gene editing can be used as a direct treatment approach or to help establish better animal models that more faithfully mimic human neurodegenerative diseases. In this manner, this technique has already shown promise in other neurological disorders, such as Huntington's disease. The purpose of this review is to examine the potential utility of CRISPR/Cas9 as a treatment option for AD by targeting specific genes including those that cause early-onset AD, as well as those that are significant risk factors for late-onset AD such as the apolipoprotein E4 (APOE4) gene.

Caspase-cleavage of tau is an early event in Alzheimer disease tangle pathology.

Neurofibrillary tangles (NFTs) are composed of abnormal aggregates of the cytoskeletal protein tau. Together with amyloid beta (Abeta) plaques and neuronal and synaptic loss, NFTs constitute the primary pathological hallmarks of Alzheimer disease (AD). Recent evidence also suggests that caspases are activated early in the progression of AD and may play a role in neuronal loss and NFT pathology. Here we demonstrate that tau is cleaved at D421 (DeltaTau) by executioner caspases. Following caspase-cleavage, DeltaTau facilitates nucleation-dependent filament formation and readily adopts a conformational change recognized by the early pathological tau marker MC1. DeltaTau can be phosphorylated by glycogen synthase kinase-3beta and subsequently recognized by the NFT antibody PHF-1. In transgenic mice and AD brains, DeltaTau associates with both early and late markers of NFTs and is correlated with cognitive decline. Additionally, DeltaTau colocalizes with Abeta(1-42) and is induced by Abeta(1-42) in vitro. Collectively, our data imply that Abeta accumulation triggers caspase activation, leading to caspase-cleavage of tau, and that this is an early event that may precede hyperphosphorylation in the evolution of AD tangle pathology. These results suggest that therapeutics aimed at inhibiting tau caspase-cleavage may prove beneficial not only in preventing NFT formation, but also in slowing cognitive decline.

Nuclear Localization of Apolipoprotein E4: A New Trick for an Old Protein.

One of the most important genetic risk factors for late-onset Alzheimer's Disease (AD) is harboring the ApoE4 allele. Much is known regarding the functions of the ApoE4 protein including cholesterol transport in the CNS and a critical role in clearing beta-amyloid deposits in the AD brain. However, recent studies demonstrating the nuclear localization suggest a novel function beyond the classical known actions of ApoE4. The purpose of the current review is to examine how this secreted protein traffics to the nucleus and to discuss possible outcomes of nuclear localization in the CNS. It is suggested that proteolytic fragmentation of ApoE4 is a key step leading to nuclear localization and the outcome of this event is to initiate transcription of various genes involved in inflammation and cell death. Therefore, the nuclear localization and induction of gene expression may provide a link between harboring the ApoE4 allele and enhanced dementia risk observed in AD.

Nuclear uptake of an amino-terminal fragment of apolipoprotein E4 promotes cell death and localizes within microglia of the Alzheimer's disease brain.

Although harboring the apolipoprotein E4 (APOE4) allele is a well known risk factor in Alzheimer's disease (AD), the mechanism by which it contributes to disease risk remains elusive. To investigate the role of proteolysis of apoE4 as a potential mechanism, we designed and characterized a site-directed cleavage antibody directed at position D151 of the mature form of apoE4 and E3. Characterization of this antibody indicated a high specificity for detecting synthesized recombinant proteins corresponding to the amino acid sequences 1-151 of apoE3 and E4 that would generate the 17 kDa (p17) fragment. In addition, this antibody also detected a ~17 kDa amino-terminal fragment of apoE4 following incubation with collagenase and matrix metalloproteinase-9 (MMP-9), but did not react with full-length apoE4. Application of this amino-terminal apoE cleavage-fragment (nApoECFp17) antibody, revealed nuclear labeling within glial cells and labeling of a subset of neurofibrillary tangles in the human AD brain. A quantitative analysis indicated that roughly 80% of labeled nuclei were microglia. To confirm these findings, cultured BV2 microglia cells were incubated with the amino-terminal fragment of apoE4 corresponding to the cleavage site at D151. The results indicated efficient uptake of this fragment and trafficking to the nucleus that also resulted in significant cell death. In contrast, a similarly designed apoE3 fragment showed no toxicity and primarily localized within the cytoplasm. These data suggest a novel cleavage event by which apoE4 is cleaved by the extracellular proteases, collagenase and MMP-9, generating an amino-terminal fragment that is then taken up by microglia, traffics to the nucleus and promotes cell death. Collectively, these findings provide important mechanistic insights into the mechanism by which harboring the APOE4 allele may elevate dementia risk observed in AD.