LRRC25 expression during physiological aging and in mouse models of Alzheimer's disease and iPSC-derived neurons.

The leucine-rich repeat-containing protein 25 (LRRC25) is relatively a novel protein with no information on its role in neuronal or brain function. A recent study suggested LRRC25 is a potential risk factor for Alzheimer's disease (AD). As a first step to understanding LRRC25's role in the brain and AD, we found LRRC25 is expressed in both cell membranes and cytoplasm in a punctuate appearance in astrocytes, microglia, and neurons in cell lines as well as mouse brain. We also found that LRRC25 expression is both age- and brain region-dependent and that 1-day-old (1D) pups expressed the least amount of LRRC25 protein compared to adult ages. In the APΔE9 mice, immunoblot quantified LRRC25 protein levels were increased by 166% (**p < 0.01) in the cortex (CX) and by 215% (***p < 0.001) in the hippocampus (HP) relative to wild-type (WT) controls. Both the brainstem (BS) and cerebellum (CB) showed no significant alterations. In the 3xTg mice, only CX showed an increase of LRRC25 protein by 91% (*p < 0.05) when compared to WT controls although the increased trend was noted in the other brain regions. In the AD patient brains also LRRC25 protein levels were increased by 153% (***p < 0.001) when compared to normal control (NC) subjects. Finally, LRRC25 expression in the iPSC-derived neurons quantified by immunofluorescence was increased by 181% (**p < 0.01) in AD-derived neurons when compared to NC-derived neurons. Thus increased LRRC25 protein in multiple models of AD suggests that LRRC25 may play a pathogenic role in either Aß or tau pathology in AD. The mechanism for the increased levels of LRRC25 in AD is unknown at present, but a previous study showed that LRRC25 levels also increase during neonatal hypoxic-ischemia neuronal damage. Based on the evidence that autophagy is highly dysregulated in AD, the increased LRRC25 levels may be due to decreased autophagic degradation of LRRC25. Increased LRRC25 in turn may regulate the stability or activity of key enzymes involved in either Aß or hyperphosphorylated tau generation and thus may contribute to increased plaques and neurofibrillary tangles.

Identification of binding partners that facilitate membrane-type 5 matrix metalloproteinase (MT5-MMP) processing of amyloid precursor protein.

One of the pathological hallmarks of Alzheimer's disease (AD) is the presence of extracellular deposits of amyloid beta (Aß) peptide. In addition to Aß as the core component of the amyloid plaque, the amyloid precursor protein (APP) processing fragment Aß was also found accumulated around the plaque. The APPη pathway, mainly mediated by membrane-type 5 matrix metalloproteinase (MT5-MMP), represents an important factor in AD pathogenesis. The proamyloidogenic features of MT5-MMP could result from interactions with APP when trafficking between organelles, so determination of the location within the cell of APPη cleavage and interacting proteins of MT5-MMP affecting this process will be of priority in understanding the role of MT5-MMP in AD. In the present study, MT5-MMP was found to be located in the nucleus, cytosol, and cytosolic subcellular granules of CHO cells that stably expressed wild-type human APP751. MT5-MMP fusion proteins were constructed that could localize enzyme production in the Golgi apparatus, endosome, ER, mitochondria, or plasma membrane. The fusion proteins significantly increased sAPPη when directed to the endosome, Golgi apparatus, plasma membrane, or mitochondria. Since the C-terminal region of MT5-MMP is responsible for its intracellular location and trafficking, this domain was used as the bait in a yeast two-hybrid screen to identify MT5-MMP protein partners in a human brain cDNA library. Identified binding partners included N4BP2L1, TMX3, EIG121, bridging Integrator 1 (BIN1), RUFY4, HTRA1, and TMEM199. The binding of N4BP2L1, EIG121, BIN1, or TMX3 to MT5-MMP resulted in the most significant increase in sAPPη production. Thus, the action of MT5-MMP on APP occurs in multiple locations within the cell and is facilitated by site-specific binding partners.

Dementia exacerbates periodontal bone loss in females.

BACKGROUND: Periodontitis is a chronic inflammatory disease defined by the pathologic loss of the periodontal ligament and alveolar bone in relation to aging. Although clinical cohort studies reported that periodontitis is significantly elevated in males compared to females, emerging evidence indicates that females with dementia are at a greater risk for periodontitis and decreased alveolar bone. OBJECTIVE: This study aimed to evaluate whether dementia is a potential sex-dependent risk factor for periodontal bone loss using an experimental model of periodontitis induced in the triple transgenic (3x-Tg) dementia-like mice and clinical samples collected from senior 65 plus age patients with diagnosed dementia. MATERIALS AND METHODS: We induced periodontitis in dementia-like triple-transgenic (3x-Tg) male and female mice and age-matched wild-type (WT) control mice by ligature placement. Then, alveolar bone loss and osteoclast activity were evaluated using micro-CT and in situ imaging assays. In addition, we performed dental examinations on patients with diagnosed dementia. Finally, dementia-associated Aß42 and p-Tau (T181) and osteoclastogenic receptor activator of nuclear factor kappa-Β ligand (RANKL) in gingival crevicular fluid (GCF) collected from mice and clinical samples were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Alveolar bone loss and in situ osteoclast activity were significantly elevated in periodontal lesions of 3x-Tg females but not males, compared to wild-type control mice. In addition, we also observed that the probing pocket depth (PPD) was also significantly elevated in female patients with dementia. Using ELISA assay, we observed that females had elevated levels of osteoclastogenic RANKL and dementia-associated Aß42 and p-Tau (T181) in the GCF collected from experimental periodontitis lesions and clinical samples. CONCLUSION: Altogether, we demonstrate that females with dementia have an increased risk for periodontal bone loss compared to males.

Novel Alzheimer risk factor IQ motif containing protein K is abundantly expressed in the brain and is markedly increased in patients with Alzheimer's disease.

Alzheimer's disease (AD) is complex and highly heterogeneous. Less than 10% of AD cases are early-onset (EOAD) caused by autosomal dominantly inherited mutations in amyloid precursor protein (APP), presenilin 1 (PS1), or presenilin 2 (PS2), each of which can increase Aß generation and, thus, amyloid plaques. The remaining 90% of cases of AD are late-onset (LOAD) or sporadic. Intense research efforts have led to identification of many genes that increase the risk of AD. An IQ motif containing protein K (IQCK) was recently identified by several investigators as an Alzheimer's disease risk gene. However, how IQCK increases AD risk is completely unknown. Since IQCK is a novel gene, there is limited information on its physiological characterization. To understand its role in AD, it is first important to determine its subcellular localization, whether and where it is expressed in the brain, and what type of brain cells express the IQCK protein. Therefore, in this study, we show by immunocytochemical (ICC) staining that IQCK is expressed in both the nucleus and the cytoplasm of SH-SY5Y neuroblastoma cells as well as HeLa cells but not in either HMC3 microglial or CHO cells. By immunohistochemistry (IHC), we also show that IQCK is expressed in both mouse and human neurons, including neuronal processes in vivo in the mouse brain. IHC data also show that the IQCK protein is widely expressed throughout the mouse brain, although regional differences were noted. IQCK expression was highest in the brainstem (BS), followed by the cerebellum (CB) and the cortex (CX), and it was lowest in the hippocampus (HP). This finding was consistent with data from an immunoblot analysis of brain tissue homogenates. Interestingly, we found IQCK expression in neurons, astrocytes, and oligodendrocytes using cell-specific antibodies, but IQCK was not detected in microglial cells, consistent with negative in vitro results in HMC3 cells. Most importantly, we found that actin-normalized IQCK protein levels were increased by 2 folds in AD brains relative to normal control (NC) brains. Furthermore, the IQCK protein was found in amyloid plaques, suggesting that IQCK may play a pathogenic role in either Aß generation or amyloid plaque deposition in AD.

Autophagy-Dependent Increased ADAM10 Mature Protein Induced by TFEB Overexpression Is Mediated Through PPARα.

Nonamyloidogenic processing of amyloid precursor protein (APP) by augmenting ADAM10 is a promising therapeutic strategy for Alzheimer's disease (AD). Therefore identification of molecular pathways that regulate ADAM10 expression is crucial. Autophagy is strongly dysregulated in AD, and TFEB was recently shown to be a master regulator of autophagy-lysosome pathway (ALP). Here, we report that TFEB expression in HeLa cells increased ADAM10 mature form by 72% (p < 0.01, n = 4), while TFEB knockdown by CRISPR strategy reduced ADAM10 mature form by 36% (p < 0.05, n = 4). Autophagy inhibition by 3-methyladenine (3-MA), but not bafilomycin A1 (BAF1), reduced ADAM10 mature form by 49% (p < 0.05, n = 4) in the TFEB expressing HeLa cells. Autophagy activation by 3 h of starvation increased ADAM10 to 91% (p < 0.001, n = 6) relative to 51% (p < 0.01, n = 6) in the nutrient-fed cells. Further, siRNAs targeted against PPARα in HeLa cells decreased ADAM10 levels by 28% (p < 0.05, n = 6) relative to the cells treated with scrambled siRNAs. Further, incubation of EGFP-TFEB expressing HeLa cells with PPARα antagonist, but not PPARß or PPARγ antagonists, prevented TFEB-induced increase in ADAM10 levels. Importantly, flag-TFEB expression in the brain also increased ADAM10 by 60% (p < 0.05, n = 3) in the cortical and 34% (p < 0.001, n = 3) in the hippocampal homogenates. ADAM10 activity also increased by 57% (p < 0.01, n = 3) in the HeLa cells. Finally, TFEB-induced ADAM10 potentiation led to increased secretion of sAPPα by 154% (p < 0.001, n = 3) in the cortex and 62% (p < 0.001, n = 3) in the hippocampus. Thus, TFEB expression enhances nonamyloidogenic processing of APP. In conclusion, TFEB expression induces ADAM10 in an autophagy-dependent manner through PPARα.

SARS-CoV-2 Infection in the Central and Peripheral Nervous System-Associated Morbidities and Their Potential Mechanism.

The recent outbreak of SARS-CoV-2 infections that causes coronavirus-induced disease of 2019 (COVID-19) is the defining and unprecedented global health crisis of our time in both the scale and magnitude. Although the respiratory tract is the primary target of SARS-CoV-2, accumulating evidence suggests that the virus may also invade both the central nervous system (CNS) and the peripheral nervous system (PNS) leading to numerous neurological issues including some serious complications such as seizures, encephalitis, and loss of consciousness. Here, we present a comprehensive review of the currently known role of SARS-CoV-2 and identify all the neurological problems reported among the COVID-19 case reports throughout the world. The virus might gain entry into the CNS either through the trans-synaptic route via the olfactory neurons or through the damaged endothelium in the brain microvasculature using the ACE2 receptor potentiated by neuropilin-1 (NRP-1). The most critical of all symptoms appear to be the spontaneous loss of breathing in some COVID-19 patients. This might be indicative of a dysfunction within the cardiopulmonary regulatory centers in the brainstem. These pioneering studies, thus, lay a strong foundation for more in-depth basic and clinical research required to confirm the role of SARS-CoV-2 infection in neurodegeneration of critical brain regulatory centers.

Potential Role of Phosphoglycerol Dihydroceramide Produced by Periodontal Pathogen Porphyromonas gingivalis in the Pathogenesis of Alzheimer's Disease.

Background: Among different types of sphingolipids produced by human cells, the possible engagement of ceramide species in the pathogenesis of Alzheimer's disease (AD) has attracted recent attention. While ceramides are primarily generated by de novo synthesis in mammalian cells, only a limited number of bacterial species, produce ceramides, including phosphoglycerol dihydroceramide (PGDHC) that is produced by the key periodontal pathogen Porphyromonas gingivalis. Emerging evidence indicates that virulence factors produced by P. gingivalis, such as lipopolysaccharide and gingipain, may be engaged in the initiation and/or progression of AD. However, the potential role of PGDHC in the pathogenesis of AD remains unknown. Therefore, the aim of this study was to evaluate the influence of PGDHC on hallmark findings in AD. Material and Methods: CHO-7WD10 and SH-SY-5Y cells were exposed to PGDHC and lipopolysaccharide (LPS) isolated from P. gingivalis. Soluble Aß42 peptide, amyloid precursor protein (APP), phosphorylated tau and senescence-associated secretory phenotype (SASP) factors were quantified using ELISA and Western blot assays. Results: Our results indicate that P. gingivalis (Pg)-derived PGDHC, but not Pg-LPS, upregulated secretion of soluble Aß42 peptide and expression of APP in CHO-7WD10 cells. Furthermore, hyperphosphorylation of tau protein was observed in SH-SY-5Y cells in response to PGDHC lipid. In contrast, Pg-LPS had little, or no significant effect on the tau phosphorylation induced in SH-SY-5Y cells. However, both PGDHC and Pg-LPS contributed to the senescence of SH-SY5Y cells as indicated by the production of senescence-associated secretory phenotype (SASP) markers, including beta-galactosidase, cathepsin B (CtsB), and pro-inflammatory cytokines TNF-α, and IL-6. Additionally, PGDHC diminished expression of the senescence-protection marker sirtuin-1 in SH-SY-5Y cells. Conclusions: Altogether, our results indicate that P. gingivalis-derived PGDHC ceramide promotes amyloidogenesis and hyperphosphorylation, as well as the production of SASP factors. Thus, PGDHC may represent a novel class of bacterial-derived virulence factors for AD associated with periodontitis.

Nanoparticle-mediated approaches for Alzheimer's disease pathogenesis, diagnosis, and therapeutics.

Alzheimer's disease (AD) is an irreversible and progressive neurodegenerative disorder manifested by memory loss and cognitive impairment. Deposition of the amyloid ß plaques has been identified as the most common AD pathology; however, the excessive accumulation of phosphorylated or total tau proteins, reactive oxygen species, and higher acetylcholinesterase activity are also strongly associated with Alzheimer's dementia. Several therapeutic approaches targeting these pathogenic mechanisms have failed in clinical or preclinical trials, partly due to the limited bioavailability, poor cell, and blood-brain barrier penetration, and low drug half-life of current regimens. The nanoparticles (NPs)-mediated drug delivery systems improve drug solubility and bioavailability, thus renders as superior alternatives. Moreover, NPs-mediated approaches facilitate multiple drug loading and targeted drug delivery, thereby increasing drug efficacy. However, certain NPs can cause acute toxicity damaging cellular and tissue architecture, therefore, NP material should be carefully selected. In this review, we summarize the recent NPs-mediated studies that exploit various pathologic mechanisms of AD by labeling, identifying, and treating the affected brain pathologies. The disadvantages of the select NP-based deliveries and the future aspects will also be discussed.

Correction: RanBP9 Overexpression Accelerates Loss of Pre and Postsynaptic Proteins in the APΔE9 Transgenic Mouse Brain.

[This corrects the article DOI: 10.1371/journal.pone.0085484.].

TFEB-mediated Enhancement of the Autophagy-lysosomal Pathway Dually Modulates the Process of Amyloid ß-Protein Generation in Neurons.

Abnormalities of the autophagy-lysosomal pathway (ALP) have been implicated in the pathology of Alzheimer's disease (AD). Activation of TFEB (transcription factor EB), a master regulator of the ALP, leads to ALP facilitation. The present study sought to clarify whether TFEB-mediated ALP facilitation influences the process of amyloid ß-protein (Aß) generation in neurons. TFEB was overexpressed in mature rat primary cortical neurons via recombinant adenoviruses, without (basal conditions) or with co-overexpression of wild-type amyloid precursor protein (APP) or its ß-C-terminal fragment (ß-CTF). We confirmed that TFEB overexpression upregulated the lysosomal proteins, cathepsin D and LAMP-1. In TFEB-expressing neurons, protein levels of ADAM10 were profoundly increased, whereas those of APP, BACE1, or γ-secretase complex proteins were unaffected. However, TFEB did not affect ADAM10 mRNA levels. TFEB overexpression had different effects on Aß production depending on the expression level of APP or ß-CTF: TFEB slightly decreased Aß secretion under basal conditions; clearly increased α-CTF levels and marginally increased ß-CTF levels with modest increases in secreted Aß in APP-expressing neurons; and caused a remarkable increase in ß-CTF levels with a significant increase in secreted Aß in ß-CTF-expressing neurons. Inhibition of proteasomes, but not lysosomes, markedly increased ß-CTF levels in ß-CTF-expressing neurons. These results collectively indicate that TFEB modulates Aß production not only by increasing α-secretase processing of APP through ADAM10 upregulation but also by augmenting ß-CTF levels possibly via altered proteasome-mediated catabolism. Thus, TFEB-mediated ALP enhancement appears to have dual, but opposite, effects on Aß production in neurons.

The neurotoxicity of amyloid ß-protein oligomers is reversible in a primary neuron model.

Alzheimer's disease (AD) is characterized by the accumulation of extracellular amyloid ß-protein (Aß) and intracellular hyperphosphorylated tau proteins. Recent evidence suggests that soluble Aß oligomers elicit neurotoxicity and synaptotoxicity, including tau abnormalities, and play an initiating role in the development of AD pathology. In this study, we focused on the unclarified issue of whether the neurotoxicity of Aß oligomers is a reversible process. Using a primary neuron culture model, we examined whether the neurotoxic effects induced by 2-day treatment with Aß42 oligomers (Aß-O) are reversible during a subsequent 2-day withdrawal period. Aß-O treatment resulted in activation of caspase-3 and eIF2α, effects that were considerably attenuated following Aß-O removal. Immunocytochemical analyses revealed that Aß-O induced aberrant phosphorylation and caspase-mediated cleavage of tau, both of which were mostly reversed by Aß-O removal. Furthermore, Aß-O caused intraneuronal dislocation of ß-catenin protein and a reduction in its levels, and these alterations were partially reversed upon Aß-O withdrawal. The dislocation of ß-catenin appeared to reflect synaptic disorganization. These findings indicate that removal of extracellular Aß-O can fully or partially reverse Aß-O-induced neurotoxic alterations in our neuron model. Accordingly, we propose that the induction of neurotoxicity by Aß oligomers is a reversible process, which has important implications for the development of AD therapies.

Transcription Factor EB Is Selectively Reduced in the Nuclear Fractions of Alzheimer's and Amyotrophic Lateral Sclerosis Brains.

Multiple studies suggest that autophagy is strongly dysregulated in Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS), as evidenced by accumulation of numerous autophagosomes, lysosomes with discontinuous membranes, and aggregated proteins in the patients' brains. Transcription factor EB (TFEB) was recently discovered to be a master regulator of lysosome biogenesis and autophagy. To examine whether aberrant autophagy in AD and ALS is due to alterations in TFEB expression, we systematically quantified the levels of TFEB in these brains by immunoblotting. Interestingly, cytoplasmic fractions of AD brains showed increased levels of normalized (to tubulin) TFEB only at Braak stage IV (61%, p < 0.01). Most importantly, normalized (to lamin) TFEB levels in the nuclear fractions were consistently reduced starting from Braak stage IV (52%, p < 0.01), stage V (67%, p < 0.01), and stage VI (85%, p < 0.01) when compared to normal control (NC) brains. In the ALS brains also, nuclear TFEB levels were reduced by 62% (p < 0.001). These data suggest that nuclear TFEB is selectively lost in ALS as well as AD brains, in which TFEB reduction was Braak-stage-dependent. Taken together, the observed reductions in TFEB protein levels may be responsible for the widely reported autophagy defects in these disorders.

TFEB Overexpression in the P301S Model of Tauopathy Mitigates Increased PHF1 Levels and Lipofuscin Puncta and Rescues Memory Deficits.

Transcription factor EB (TFEB) was recently shown to be a master regulator of autophagy lysosome pathway. Here, we successfully generated and characterized transgenic mice overexpressing flag-TFEB. Enhanced autophagy in the flag-TFEB transgenic mice was confirmed by an increase in the cellular autophagy markers, as determined by both immunoblots and transmission electron microscopy. Surprisingly, in the flag-TFEB mice we observed increased activity of senescence-associated ß-galactosidase by ∼66% of neurons in the cortex (p < 0.001) and 73% of neurons in the hippocampus (p < 0.001). More importantly, flag-TFEB expression remarkably reduced the levels of paired-helical filament (PHF)-tau from 372% in the P301S model of tauopathy to 171% (p < 0.001) in the cortex, and from 436% to 212% (p < 0.001) in the hippocampus. Significantly, reduced levels of NeuN in the cortex (38%, p < 0.001) and hippocampus (25%, p < 0.05) of P301S mice were almost completely restored to WT levels in the P301S/flag-TFEB double-transgenic mice. Also, levels of spinophilin in both the cortex (p < 0.001) and hippocampus (p < 0.001) were restored almost to WT levels. Most importantly, the age-associated lipofuscin granules, which are generally presumed to be nondegradable, were reduced by 57% (p < 0.001) in the cortex and by 55% (p < 0.001) in the hippocampus in the double-transgenic mice. Finally, TFEB overexpression in the P301S mice markedly reversed learning deficits in terms of spatial memory (Barnes maze), as well as working and reference memories (T maze). These data suggest that the overexpression of TFEB can reliably enhance autophagy in vivo, reduce levels of PHF-tau, and thereby reverse the deposition of lipofuscin granules and memory deficits.

LRP1 Downregulates the Alzheimer's ß-Secretase BACE1 by Modulating Its Intraneuronal Trafficking

The ß-secretase called BACE1 is a membrane-associated protease that initiates the generation of amyloid ß-protein (Aß), a key event in Alzheimer's disease (AD). However, the mechanism of intraneuronal regulation of BACE1 is poorly understood. Here, we present evidence that low-density lipoprotein receptor-related protein 1 (LRP1), a multi-functional receptor, has a previously unrecognized function to regulate BACE1 in neurons. We show that deficiency of LRP1 exerts promotive effects on the protein expression and function of BACE1, whereas expression of LRP-L4, a functional LRP1 mini-receptor, specifically decreases BACE1 levels in both human embryonic kidney (HEK) 293 cells and rat primary neurons, leading to reduced Aß production. Our subsequent analyses further demonstrate that (1) both endogenous and exogenous BACE1 and LRP1 interact with each other and are colocalized in soma and neurites of primary neurons, (2) LRP1 reduces the protein stability and cell-surface expression of BACE1, and (3) LRP1 facilitates the shift in intracellular localization of BACE1 from early to late endosomes, thereby promoting lysosomal degradation. These findings establish that LRP1 specifically downregulates BACE1 by modulating its intraneuronal trafficking and stability through protein interaction and highlight LRP1 as a potential therapeutic target in AD.

COPS5 protein overexpression increases amyloid plaque burden, decreases spinophilin-immunoreactive puncta, and exacerbates learning and memory deficits in the mouse brain.

Brain accumulation of neurotoxic amyloid ß (Aß) peptide because of increased processing of amyloid precursor protein (APP), resulting in loss of synapses and neurodegeneration, is central to the pathogenesis of Alzheimer disease (AD). Therefore, the identification of molecules that regulate Aß generation and those that cause synaptic damage is crucial for future therapeutic approaches for AD. We demonstrated previously that COPS5 regulates Aß generation in neuronal cell lines in a RanBP9-dependent manner. Consistent with the data from cell lines, even by 6 months, COPS5 overexpression in APΔE9 mice (APΔE9/COPS5-Tg) significantly increased Aß40 levels by 32% (p < 0.01) in the cortex and by 28% (p < 0.01) in the hippocampus, whereas the increases for Aß42 were 37% (p < 0.05) and 34% (p < 0.05), respectively. By 12 months, the increase was even more robust. Aß40 levels increased by 63% (p < 0.001) in the cortex and by 65% (p < 0.001) in the hippocampus. Similarly, Aß42 levels were increased by 69% (p < 0.001) in the cortex and by 71% (p < 0.011) in the hippocampus. Increased Aß levels were translated into an increased amyloid plaque burden both in the cortex (54%, p < 0.01) and hippocampus (64%, p < 0.01). Interestingly, COPS5 overexpression increased RanBP9 levels in the brain, which, in turn, led to increased amyloidogenic processing of APP, as reflected by increased levels of sAPPß and decreased levels of sAPPα. Furthermore, COPS5 overexpression reduced spinophilin in both the cortex (19%, p < 0.05) and the hippocampus (20%, p < 0.05), leading to significant deficits in learning and memory skills. Therefore, like RanBP9, COPS5 also plays a pivotal role in amyloid pathology in vivo.

Substituted dithiazole piperazine benzamides as novel amyloid beta peptide reducing agents.

Alzheimer's disease is a persistent neurodegenerative disorder of elderly characterized clinically by irreversible loss of memory due to accumulation of amyloid beta peptides within the amyloid plaques. We report the parallel synthesis and screening results of diverse substituted di-thiazole piperazine benzamides. A new compound TPI-1917-49 was identified as a promising amyloid reducing agent by lowering the levels of Aß at least in two cell types and in vivo.

RanBP9 overexpression accelerates loss of dendritic spines in a mouse model of Alzheimer's disease.

We previously demonstrated that RanBP9 overexpression increased Aß generation and amyloid plaque burden, subsequently leading to robust reductions in the levels of several synaptic proteins as well as deficits in the learning and memory skills in a mouse model of Alzheimer's disease (AD). In the present study, we found striking reduction of spinophilin-immunoreactive puncta (52%, p<0.001) and spinophilin area (62.5%, p<0.001) in the primary cortical neurons derived from RanBP9 transgenic mice (RanBP9-Tg) compared to wild-type (WT) neurons. Similar results were confirmed in WT cortical neurons transfected with EGFP-RanBP9. At 6-months of age, the total spine density in the cortex of RanBP9 single transgenic, APΔE9 double transgenic and APΔE9/RanBP9 triple transgenic mice was similar to WT mice. However, in the hippocampus the spine density was significantly reduced (27%, p<0.05) in the triple transgenic mice compared to WT mice due to reduced number of thin spines (33%, p<0.05) and mushroom spines (22%, p<0.05). This suggests that RanBP9 overexpression in the APΔE9 mice accelerates loss of spines and that the hippocampus is more vulnerable. At 12-months of age, the cortex showed significant reductions in total spine density in the RanBP9 (22%, p<0.05), APΔE9 (19%, p<0.05) and APΔE9/RanBP9 (33%, p<0.01) mice compared to WT controls due to reductions in mushroom and thin spines. Similarly, in the hippocampus the total spine density was reduced in the RanBP9 (23%, p<0.05), APΔE9 (26%, p<0.05) and APΔE9/RanBP9 (39%, p<0.01) mice due to reductions in thin and mushroom spines. Most importantly, RanBP9 overexpression in the APΔE9 mice further exacerbated the reductions in spine density in both the cortex (14%, p<0.05) and the hippocampus (16%, p<0.05). Because dendritic spines are considered physical traces of memory, loss of spines due to RanBP9 provided the physical basis for the learning and memory deficits. Since RanBP9 protein levels are increased in AD brains, RanBP9 might play a crucial role in the loss of spines and synapses in AD.

AlphaLISA-based high-throughput screening assay to measure levels of soluble amyloid precursor protein α.

Activation of nonamyloidogenic processing of amyloid precursor protein (APP) has been hypothesized to be a viable approach for Alzheimer's disease drug discovery. However, until recently, the lack of HTS-compatible assay technologies precluded large scale screening efforts to discover molecules that potentiate nonamyloidogenic pathways. We have developed an HTS-compatible assay based on AlphaLISA technology that quantitatively detects soluble APPα (sAPPα), a marker of nonamyloidogenic processing of APP, released from live cells in low volume, 384-well plates. The assay exhibited good QC parameters (Z'>0.5, S/B>2). A pilot screen of 801 compounds yielded a novel chemotype that increased the release of sAPPα 2-fold at 5µM. These results suggest that the AlphaLISA-based HTS assay is robust and sensitive and can be used to screen large compound collections to discover molecules that potentiate the release of sAPPα. Additionally, we demonstrated that increase of APP processing by nonamyloidogenic pathways will result in decrease of release of amyloidogenic Aß40 fragments.

RanBP9 overexpression accelerates loss of pre and postsynaptic proteins in the APΔE9 transgenic mouse brain.

There is now compelling evidence that the neurodegenerative process in Alzheimer's disease (AD) begins in synapses. Loss of synaptic proteins and functional synapses in the amyloid precursor protein (APP) transgenic mouse models of AD is well established. However, what is the earliest age at which such loss of synapses occurs, and whether known markers of AD progression accelerate functional deficits is completely unknown. We previously showed that RanBP9 overexpression leads to robustly increased amyloid ß peptide (Aß) generation leading to enhanced amyloid plaque burden in a mouse model of AD. In this study we compared synaptic protein levels among four genotypes of mice, i.e., RanBP9 single transgenic (Ran), APΔE9 double transgenic (Dbl), APΔE9/RanBP9 triple transgenic (Tpl) and wild-type (WT) controls. We found significant reductions in the levels of synaptic proteins in both cortex and hippocampus of 5- and 6-months-old but not 3- or 4-months-old mice. Specifically, at 5-months of age, rab3A was reduced in the triple transgenic mice only in the cortex by 25% (p<0.05) and gap43 levels were reduced only in the hippocampus by 44% (p<0.01) compared to wild-type (WT) controls. Interestingly, RanBP9 overexpression in the Tpl mice reduced gap43 levels by a further 31% (p<0.05) compared to APΔE9 mice. RanBP9 also further decreased the levels of drebrin in the hippocampus by 32% (p<0.01) and chromogranin in the cortex by 24% (p<0.05) compared to APΔE9 mice. At 6-months of age, RanBP9 expression in the cortex led to further reduction of rab3A by 30% (p<0.05) and drebrin by 38% (p<0.01) compared to APΔE9 mice. RanBP9 also increased Aß oligomers in the cortex at 6 months. Similarly, in the hippocampus, RanBP9 expression further reduced rab3A levels by 36% (p<0.01) and drebrin levels by 33% (p<0.01). Taken together these data suggest that RanBP9 overexpression accelerates loss of synaptic proteins in the mouse brain.