Mouse Brain Extracts Research Articles

Discovery of a novel Xanthone derivative P24 for anti-AD via targeting sTGFBR3

Soluble transforming growth factor beta receptor 3 (sTGFBR3) antagonist is a new focus in the research and development of Alzheimer's disease (AD) drugs. Our previous studies have identified sTGFBR3 as a promising new target for AD, with few targeted antagonists identified. In this study, we performed structural modeling of sTGFBR3 using AlphaFold2, followed by high-throughput virtual screening and surface plasmon resonance assays. which collectively identified Xanthone as potential compounds for targeting sTGFBR3. After optimizing the sTGFBR3-Xanthone complex using molecular dynamics (MD) simulations, we prepared a series of novel Xanthone derivatives and evaluated their anti-inflammatory activity, toxicity, and structure-activity relationship in BV2 cell model induced by lipopolysaccharides (LPS) or APP/PS1/tau mouse brain extract (BE). Several derivatives with the most potent anti-inflammatory activity were tested for blood-brain barrier permeability and sTGFBR3 affinity. Derivative P24, selected for its superior properties, was further evaluated in vitro. The results indicated that P24 increased the activation of TGF-β signaling and decreased the activation of IκBα/NF-κB signaling by targeting sTGFBR3, thereby regulating the inflammation-phagocytosis balance in microglia. Moreover, the low acute toxicity, long half-life, and low plasma clearance of P24 suggest that it can be sustained in vivo. This property may render P24 a more effective treatment modality for chronic diseases, particularly AD. The study demonstrates P24 serve as potential novel candidates for the treatment of AD via antagonizing sTGFBR3.

Nanorod-associated plasmonic circular dichroism monitors the handedness and composition of α-synuclein fibrils from Parkinson's disease models and post-mortem brain.

Human full-length (fl) αSyn fibrils, key neuropathological hallmarks of Parkinson's disease (PD), generate intense optical activity corresponding to the surface plasmon resonance of interacting gold nanorods. Herein, we analysed fibril-enriched protein extracts from mouse and human brain samples as well as from SK-N-SH cell lines with or without human fl and C-terminally truncated (Ctt) αSyn overexpression and exposed them to αSyn monomers, recombinant fl αSyn fibrils or Ctt αSyn fibrils. In vitro-generated human recombinant fl and Ctt αSyn fibrils and fibrils purified from SK-N-SH cells with fl or Ctt αSyn overexpression were also analysed using transmission electron microscopy (TEM) to gain insights into the nanorod-fibril complexes. We found that under the same experimental conditions, bisignate circular dichroism (CD) spectra of Ctt αSyn fibrils exhibited a blue-wavelength shift compared to that of fl αSyn fibrils. TEM results supported that this could be attributed to the different properties of nanorods. In our experimental conditions, fibril-enriched PD brain extract broadened the longitudinal surface plasmonic band with a bisignate CD couplet centred corresponding to the absorption band maximum. Plasmonic CD (PCD) couplets of in vivo- and in vitro-generated fibrils displayed sign reversal, suggesting their opposite handedness. Moreover, the incubation of in vitro-generated human recombinant fl αSyn fibrils in mouse brain extracts from αSyn null mice resulted in PCD couplet inversion, indicating that the biological environment may shape the handedness of αSyn fibrils. These findings support that nanorod-based PCD can provide useful information on the composition and features of αSyn fibrils from biological materials.

Spatacsin regulates directionality of lysosome trafficking by promoting the degradation of its partner AP5Z1.

The endoplasmic reticulum (ER) forms contacts with the lysosomal compartment, regulating lysosome positioning and motility. The movements of lysosomes are controlled by the attachment of molecular motors to their surface. However, the molecular mechanisms by which ER controls lysosome dynamics are still elusive. Here, using mouse brain extracts and mouse embryonic fibroblasts, we demonstrate that spatacsin is an ER-resident protein regulating the formation of tubular lysosomes, which are highly dynamic. Screening for spatacsin partners required for tubular lysosome formation showed spatacsin to act by regulating protein degradation. We demonstrate that spatacsin promotes the degradation of its partner AP5Z1, which regulates the relative amount of spastizin and AP5Z1 at lysosomes. Spastizin and AP5Z1 contribute to regulate tubular lysosome formation, as well as their trafficking by interacting with anterograde and retrograde motor proteins, kinesin KIF13A and dynein/dynactin subunit p150Glued, respectively. Ultimately, investigations in polarized mouse cortical neurons in culture demonstrated that spatacsin-regulated degradation of AP5Z1 controls the directionality of lysosomes trafficking. Collectively, our results identify spatacsin as a protein regulating the directionality of lysosome trafficking.

Real-time bottom-up characterization of protein mixtures enabled by online microdroplet-assisted enzymatic digestion (MAED).

Enzymatic digestion remains one of the "rate-determining steps" in the bottom-up analysis of proteins. However, by performing digestion in microdroplets generated from electrosonic spray, the reaction could be accelerated to a timescale lower than milliseconds. Here, we describe a simple and rapid online digestion platform named online microdroplet-assisted enzymatic digestion (MAED). It involves the integration of intact protein separation with enzymatic digestion in microdroplets. Via online MAED, various protein standards, including an antibody standard, were characterized in a bottom-up manner without prior digestion, and high sequence coverages were obtained. We further extended the application of online MAED to a more complex sample, mouse brain extract, where protein identifications were successfully yielded. Compared with the conventional bottom-up approach, a more comprehensive characterization could be obtained particularly for low molecular weight proteins. In short, we provide a rapid and alternative bottom-up analysis in a top-down fashion as well as a new possibility for microdroplet chemistry.

PS-B05-10: DIABETES MELLITUS AGGRAVATES BEHAVIORAL DEFICIT AND INCREASES SITE-SPECIFIC PHOSPHORYLATION OF TAU PROTEIN IN ALZHEIMER'S DISEASE MOUSE MODEL

Objective: Numerous epidemiological studies have demonstrated that vascular risk factors including diabetes mellitus (DM) increase the risk of developing Alzheimer's disease (AD). However, the mechanisms underlying the pathological relationship between DM and AD remain largely unknown. Tau is a microtubule-associated protein that is predominantly expressed in the axons of neurons. More than 80 residues of tau can be potentially phosphorylated. Accumulation of hyperphosphorylated tau protein plays a role in neurodegeneration and cognitive impairment in patients with AD. Here, we developed a unique diabetic AD mouse model and analyzed behavioral phenotypes and biochemical changes occurring in the brain to investigate the impact of DM on tau pathology. Design and method: Tau-tg and WT mice were fed with either normal chow diet (NCD; 12% kcal fat, n = 9) or high-fat diet (HFD; 60% kcal fat, n = 10) from the age of one and a half to nine months. Metabolic parameters and behavioral phenotypes were assessed at eight months of age. Mice were then sacrificed at nine months of age and brains were analyzed histologically and biochemically. A quantitative proteomic analysis of protein phosphorylation was performed using brain extracts of tau-tg mice. Results: Tau-tg mice fed with HFD (HFD tau-tg) showed severe obesity and hyperinsulinemia compared with tau-tg mice fed with NCD (NCD tau-tg) (p < 0.01), with slight elevation of plasma glucose levels. HFD tau-tg showed exacerbation of motor impairments and decrement of habituation to novel environment. Histological analysis showed a significant decrease in the number of neurons in HFD tau-tg compared to NCD tau-tg. The amount of aggregated form of phospho-tau (Ser396) was increased in HFD tau-tg brain. Comprehensive phosphoproteomic analysis identified 11 phosphosites of tau significantly hyperphosphorylated in the brain of HFD tau-tg (p < 0.05). Conclusions: These results suggest that diabetic conditions such as obesity and hyperinsulinemia with minimal hyperglycemia exacerbate behavioral deficits in AD via hyperphosphorylation of tau protein. We identified specific phosphosites of tau involved in pathological relationship between DM and AD.

An oligomer‐directed Aβ antibody decreases synaptotoxic oligomeric Aβ in brain and improves behavioral abnormality in humanized APP knock‐in mice

AbstractBackgroundThere is strong evidence that soluble oligomers of amyloid beta protein (oAβ) help initiate the pathogenic cascade of Alzheimer’s disease (AD), which suggests therapeutic strategies targeting oAβ over monomeric or fibrillar Aβ. A new antibody, 71A1, is 1∼00‐fold more sensitive for oAβ than synthetic monomers. The material that 71A1 specifically immunoprecipitates from AD soluble brain extracts impairs synaptic function as much as does the full extract. In accord, pre‐incubating brain extracts with 71A1 neutralizes its synaptotoxicity. 71A1 has potentially unique activities against disease‐relevant oAβ, making it a novel candidate for treating AD.Methods15 mg/kg 71A1 or anti‐KLH (negative control) was administered i.p. to 45 humanized APP knock‐in mice (APPNLGF/NLGF) weekly from 8 to 20 weeks of age. Cognition was assessed by spontaneous alternation (Y‐maze). Brains were harvested at age 21‐wk for biochemical, electrophysiological and immunohistochemical analyses. Diffusible (“soaking”) extracts of the brains were prepared to measure monomeric and oligomeric Aβ using homebrew ultra‐sensitive assays. Aliquots of the same soaking extracts were used to treat wild‐type mouse hippocampal slices and measure long‐term potentiation (LTP) to assess synaptotoxicity in the APPNLGF/NLGF brains post‐treatment.Results71A1 recognized oAβ and neutralized its synaptotoxicity in APPNLGF/NLGF mouse brain extracts. 71A1 effectively treated male APPNLGF/NLGF mice, which improved cognition significantly. The antibody reduced brain Aβ (1‐42) and oAβ levels with a trend toward statistical significance. Mouse brains with less oAβ had less synaptotoxicity as assessed by LTP.ConclusionsThese results demonstrate 71A1 is an oligomer‐preferring monoclonal antibody as a therapeutic candidate for AD, decreasing brain oAβ and oAβ‐induced synaptotoxicity. We believe this provides the first proof‐of‐concept mouse trial using an antibody that has been carefully characterized as oAβ‐preferring. We observed 1) a correlation between oAβ concentration and its synaptotoxicity; and 2) that 71A1 ameliorated aspects of cognitive impairment in APPNLGF/NLGF mice by targeting oAβ.

Simiate and the focal adhesion kinase FAK1 cooperate in the regulation of dendritogenesis

Despite the crucial importance of dendritogenesis for the correct functioning of neurons, the molecular mechanisms underlying neuronal arborisation are still not well understood. Current models suggest that distinct parts and phases of dendritic development are regulated by the expression of distinct transcription factors, that are able to target the cytoskeleton. Two proteins recently implicated in dendritogenesis are the Focal Adhesion Kinase FAK1 and the Actin-binding protein Simiate. Using heterologous expression systems as well as mouse brain extracts in combination with coprecipitation assays, we show that Simiate is able to associate with FAK1. Differential centrifugation experiments further revealed the interaction to be present in cytosolic as well as nuclear fractions. Inside the nucleus though, Simiate preferentially binds to a FAK1 isoform of 80 kDa, which has previously been shown to regulate transcription factor activity. Investigating the function of both proteins in primary hippocampal cultures, we further found that FAK1 and Simiate have distinct roles in dendritogenesis: While FAK1 increases dendrite length and number, Simiate preferentially enhances growth and branching. However, if being confined to the nucleus, Simiate selectively triggers primary dendrite formation, enhancing transcription activity at the same time. Since the effect on primary dendrites is specifically re-normalized by a co-expression of FAK1 and Simiate in the nucleus, the data implies that the two proteins interact to counterbalance each other in order to control dendrite formation. Looking at the role of the cytosolic interaction of FAK1 and Simiate, we found that neurotrophin induced dendritogenesis causes a striking colocalisation of FAK1 and Simiate in dendritic growth cones, which is not present otherwise, thus suggesting that the cytosolic interaction stimulates growth cone mediated dendritogenesis in response to certain external signals. Taken together, the data show that FAK1 and Simiate exert several and distinct actions during the different phases of dendritogenesis and that these actions are related to their subcellular localisation and their interaction.

Brain Extract of Subacute Traumatic Brain Injury Promotes the Neuronal Differentiation of Human Neural Stem Cells via Autophagy.

Background: After a traumatic brain injury (TBI), the cell environment is dramatically changed, which has various influences on grafted neural stem cells (NSCs). At present, these influences on NSCs have not been fully elucidated, which hinders the finding of an optimal timepoint for NSC transplantation. Methods: Brain extracts of TBI mice were used in vitro to simulate the different phase TBI influences on the differentiation of human NSCs. Protein profiles of brain extracts were analyzed. Neuronal differentiation and the activation of autophagy and the WNT/CTNNB pathway were detected after brain extract treatment. Results: Under subacute TBI brain extract conditions, the neuronal differentiation of hNSCs was significantly higher than that under acute brain extract conditions. The autophagy flux and WNT/CTNNB pathway were activated more highly within the subacute brain extract than in the acute brain extract. Autophagy activation by rapamycin could rescue the neuronal differentiation of hNSCs within acute TBI brain extract. Conclusions: The subacute phase around 7 days after TBI in mice could be a candidate timepoint to encourage more neuronal differentiation after transplantation. The autophagy flux played a critical role in regulating neuronal differentiation of hNSCs and could serve as a potential target to improve the efficacy of transplantation in the early phase.

SH3- and actin-binding domains connect ADNP and SHANK3, revealing a fundamental shared mechanism underlying autism.

De novo heterozygous mutations in activity-dependent neuroprotective protein (ADNP) cause autistic ADNP syndrome. ADNP mutations impair microtubule (MT) function, essential for synaptic activity. The ADNP MT-associating fragment NAPVSIPQ (called NAP) contains an MT end-binding protein interacting domain, SxIP (mimicking the active-peptide, SKIP). We hypothesized that not all ADNP mutations are similarly deleterious and that the NAPV portion of NAPVSIPQ is biologically active. Using the eukaryotic linear motif (ELM) resource, we identified a Src homology 3 (SH3) domain-ligand association site in NAP responsible for controlling signaling pathways regulating the cytoskeleton, namely NAPVSIP. Altogether, we mapped multiple SH3-binding sites in ADNP. Comparisons of the effects of ADNP mutations p.Glu830synfs*83, p.Lys408Valfs*31, p.Ser404* on MT dynamics and Tau interactions (live-cell fluorescence-microscopy) suggested spared toxic function in p.Lys408Valfs*31, with a regained SH3-binding motif due to the frameshift insertion. Site-directed-mutagenesis, abolishing the p.Lys408Valfs*31 SH3-binding motif, produced MT toxicity. NAP normalized MT activities in the face of all ADNP mutations, although, SKIP, missing the SH3-binding motif, showed reduced efficacy in terms of MT-Tau interactions, as compared with NAP. Lastly, SH3 and multiple ankyrin repeat domains protein 3 (SHANK3), a major autism gene product, interact with the cytoskeleton through an actin-binding motif to modify behavior. Similarly, ELM analysis identified an actin-binding site on ADNP, suggesting direct SH3 and indirect SHANK3/ADNP associations. Actin co-immunoprecipitations from mouse brain extracts showed NAP-mediated normalization of Shank3-Adnp-actin interactions. Furthermore, NAP treatment ameliorated aberrant behavior in mice homozygous for the Shank3 ASD-linked InsG3680 mutation, revealing a fundamental shared mechanism between ADNP and SHANK3.

Implementation of a method for analysis of Gi/o‐coupled orphan GPCR signaling

G‐protein coupled receptors (GPCRs) are currently the target of almost 30% of the marketed drugs. However, only a small percentage of the whole GPCR repertoire is well characterized for both endogenous ligands and signaling pathways, with ~100 GPCRs still considered orphans (oGPCRs). Nonetheless, oGPCRs are increasingly recognized for their essential pathophysiological roles. This pool of uncharacterized GPCRs has therefore a considerable untapped potential as new pharmacological targets. Scientists exploited several indirect approaches to discover new ligand‐receptor pairs and characterize receptor‐signaling couplings, however in the last few years the number of deorphanized GPCRs has not grown as expected. According to the “GPCR database” almost two thirds of GPCRs are coupled to Gi/o/z proteins, for which currently available assays display low sensitivity. To address part of this problem, we recently used different reporter systems in combination with Gα‐protein chimeras to uncover Gα‐protein coupling by measuring the constitutive activity of a library of oGPCRs expressed in a heterologous system. Using this assay we identified 8 out of 19 oGPCRs tested as coupled to Gi/o/z proteins. In the present project, we aim at setting up a new in vitro high‐throughput screening assay for Gi/o/z‐coupled oGPCRs, combining Gi/o/z‐protein chimeras and cAMP measurements using GloSensor. Our preliminary data obtained studying traditional Gi/o/z‐coupled receptors suggest that expression of the GsGz chimera in combination with Pertussis Toxin (PTX) results in an assay displaying the highest sensitivity and signal amplitude. Considering the high expression of many oGPCRs in the brain, we hypothesized that their endogenous ligands would be also localized there. We therefore tested the activation of transfected oGPCRs using our optimized system by mouse brain extract, as a mixture of candidate ligands. The success of this preliminary analysis validates our approach for future screening purposes. Overall, implementation of this method for screening medium to large libraries of molecules would allow to deorphanize oGPCRs, providing new insights in GPCR pharmacology and the discovery of new ligand‐receptor pairs.

The Cell Adhesion Molecule L1 Interacts with Methyl CpG Binding Protein 2 via Its Intracellular Domain.

Cell adhesion molecule L1 regulates multiple cell functions, and L1 deficiency is linked to several neural diseases. Recently, we have identified methyl CpG binding protein 2 (MeCP2) as a potential binding partner of the intracellular L1 domain. By ELISA we show here that L1’s intracellular domain binds directly to MeCP2 via the sequence motif KDET. Proximity ligation assay with cultured cerebellar and cortical neurons suggests a close association between L1 and MeCP2 in nuclei of neurons. Immunoprecipitation using MeCP2 antibodies and nuclear mouse brain extracts indicates that MeCP2 interacts with an L1 fragment of ~55 kDa (L1−55). Proximity ligation assay indicates that metalloproteases, β-site of amyloid precursor protein cleaving enzyme (BACE1) and ɣ-secretase, are involved in the generation of L1−55. Reduction in MeCP2 expression by siRNA decreases L1-dependent neurite outgrowth from cultured cortical neurons as well as the migration of L1-expressing HEK293 cells. Moreover, L1 siRNA, MeCP2 siRNA, or a cell-penetrating KDET-containing L1 peptide leads to reduced levels of myocyte enhancer factor 2C (Mef2c) mRNA and protein in cortical neurons, suggesting that the MeCP2/L1 interaction regulates Mef2c expression. Altogether, the present findings indicate that the interaction of the novel fragment L1−55 with MeCP2 affects L1-dependent functions, such as neurite outgrowth and neuronal migration.

Conformation-Dependent Reversible Interaction of Ca2+/Calmodulin-Dependent Protein Kinase Kinase with an Inhibitor, TIM-063.

Ca2+/calmodulin-dependent protein kinase kinase (CaMKK), a Ca2+/CaM-dependent enzyme that phosphorylates and activates multifunctional kinases, including CaMKI, CaMKIV, protein kinase B/Akt, and 5'AMP-activated protein kinase, is involved in various Ca2+-signaling pathways in cells. Recently, we developed an ATP-competitive CaMKK inhibitor, TIM-063 (2-hydroxy-3-nitro-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one, Ohtsuka et al. Biochemistry 2020, 59, 1701-1710). To gain mechanistic insights into the interaction of CaMKK with TIM-063, we prepared TIM-063-coupled sepharose (TIM-127-sepharose) for association/dissociation analysis of the enzyme/inhibitor complex. CaMKKα/β in transfected COS-7 cells and in mouse brain extracts specifically bound to TIM-127-sepharose and dissociated following the addition of TIM-063 in a manner similar to that of recombinant GST-CaMKKα/β, which could bind to TIM-127-sepharose in a Ca2+/CaM-dependent fashion and dissociate from the sepharose following the addition of TIM-063 in a dose-dependent manner. In contrast to GST-CaMKKα, GST-CaMKKβ was able to weakly bind to TIM-127-sepharose in the presence of EGTA, probably due to the partially active conformation of recombinant GST-CaMKKβ without Ca2+/CaM-binding. These results suggested that the regulatory domain of CaMKKα prevented the inhibitor from interacting with the catalytic domain as the GST-CaMKKα mutant (residues 126-434) lacking the regulatory domain (residues 438-463) interacted with TIM-127-sepharose regardless of the presence or absence of Ca2+/CaM. Furthermore, CaMKKα bound to TIM-127-sepharose in the presence of Ca2+/CaM completely dissociated from TIM-127-sepharose following the addition of excess EGTA. These results indicated that TIM-063 interacted with and inhibited CaMKK in its active state but not in its autoinhibited state and that this interaction is likely reversible, depending on the concentration of intracellular Ca2+.

Hydrophilic arginine-functionalized mesoporous polydopamine-graphene oxide composites for glycopeptides analysis

Considering the importance of glycopeptides in the clinical diagnosis of cancer and some serious diseases, the identification of glycopeptides from complex biological samples has attracted considerable attention. Effective pre-enrichment before mass spectrometry analysis plays an important role. In this work, a kind of hydrophilic two-dimensional composites (denoted as GO@MPDA@Arg) based on mesoporous polydopamine-graphene oxide were used to selectively enrich glycopeptides in biological samples. The mesoporous polydopamine (MPDA) layer self-assembled with template Pluronic F127 provided more binding sites to load arginine, and bound arginine enhanced the hydrophilicity of the material. As a result, GO@MPDA@Arg composites exhibited excellent enrichment performance for glycopeptides, containing good selectivity (IgG digests : BSA digests = 1:50, molar ratio), low detection limit for IgG digests (10 fmol μL−1), high loading capacity for IgG digests (200 μg mg−1), and good size exclusion (IgG digests : IgG : BSA = 1:100:100, mass ratio). In addition, mouse brain tissue was selected as the actual biological sample to further study the enrichment effect of GO@MPDA@Arg composites. In three parallel experiments, a total of 401 glycopeptides belonging to 233 glycoproteins were enriched from 200 μg digestion of mouse brain extract. The enrichment results demonstrate that GO@MPDA@Arg composites have application potential for glycopeptides enrichment in protein post-translational modification research.

Unbiased high-content screening reveals Aβ- and tau-independent synaptotoxic activities in human brain homogenates from Alzheimer's patients and high-pathology controls.

Alzheimer’s disease (AD) is tightly correlated with synapse loss in vulnerable brain regions. It is assumed that specific molecular entities such as Aβ and tau cause synapse loss in AD, yet unbiased screens for synaptotoxic activities have not been performed. Here, we performed size exclusion chromatography on soluble human brain homogenates from AD cases, high pathology non-demented controls, and low pathology age-matched controls using our novel high content primary cultured neuron-based screening assay. Both presynaptic and postsynaptic toxicities were elevated in homogenates from AD cases and high pathology non-demented controls to a similar extent, with more modest synaptotoxic activities in homogenates from low pathology normal controls. Surprisingly, synaptotoxic activities were found in size fractions peaking between the 17–44 kDa size standards that did not match well with Aβ and tau immunoreactive species in these homogenates. The fractions containing previously identified high molecular weight soluble amyloid beta aggregates/”oligomers” were non-toxic in this assay. Furthermore, immunodepletion of Aβ and tau did not reduce synaptotoxic activity. This result contrasts with previous findings involving the same methods applied to 3xTg-AD mouse brain extracts. The nature of the synaptotoxic species has not been identified. Overall, our data indicates one or more potential Aβ and tau independent synaptotoxic activities in human AD brain homogenates. This result aligns well with the key role of synaptic loss in the early cognitive decline and may provide new insight into AD pathophysiology.

Small, Seeding-Competent Huntingtin Fibrils Are Prominent Aggregate Species in Brains of zQ175 Huntington's Disease Knock-in Mice.

The deposition of mutant huntingtin (mHTT) protein aggregates in neurons of patients is a pathological hallmark of Huntington’s disease (HD). Previous investigations in cell-free and cell-based disease models showed mHTT exon-1 (mHTTex1) fragments with pathogenic polyglutamine (polyQ) tracts (>40 glutamines) to self-assemble into highly stable, β-sheet-rich protein aggregates with a fibrillar morphology. HD knock-in mouse models have not been extensively studied with regard to mHTT aggregation. They endogenously produce full-length mHTT with a pathogenic polyQ tract as well as mHTTex1 fragments. Here, we demonstrate that seeding-competent, fibrillar mHTT aggregates can be readily detected in brains of zQ175 knock-in HD mice. To do this, we applied a highly sensitive FRET-based protein amplification assay that is capable of detecting seeding-competent mHTT aggregate species down to the femtomolar range. Furthermore, we show that fibrillar structures with an average length of ∼200 nm can be enriched with aggregate-specific mouse and human antibodies from zQ175 mouse brain extracts through immunoprecipitations, confirming that such structures are formed in vivo. Together these studies indicate that small, fibrillar, seeding-competent mHTT structures are prominent aggregate species in brains of zQ175 mice.

A New Fluorescent Method to Detect Sulfamidase Activity in Blood, Tissue Extracts and Dried Blood Spots

Abstract Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder due to the deficient activity of sulfamidase (SGSH). Traditionally, measurement of this enzymatic activity has been performed using a fluorescently (4-MU) labeled glycoside substrate. While this substrate is inexpensive and readily available, the current method requires a 2-step procedure that is performed over 2 days. Here we report a new and simplified procedure using the 4-MU substrate. Major advantages of this assay method over the existing fluorescent method include a single step vs. 2-step procedure, an incubation time of 1 hour, and high sensitivity. The reaction is also run on UPLC equipment, which is available in most research labs and permits separation of the endogenous, autofluorescent material from the 4-MU signal. This assay method was developed using the MPS IIIA mouse model, and was validated using mouse plasma, liver and brain extracts, and dried blood spots. Human MPS IIIA skin fibroblasts and dried blood spots also were used to validate the method.

Development of monoclonal antibodies specific for the calpain‐generated ∆48 kDa calcineurin fragment, a marker of distressed astrocytes

AbstractBackgroundCalcineurin (CN) is a Ca2+/calmodulin‐dependent protein phosphatase expressed at high levels in brain. In healthy tissue, CN exists mainly as a full‐length (∼60 kDa) highly‐regulated protein involved in essential cellular functions. However, in diseased or injured tissue, CN is proteolytically converted to a constitutively active fragment that has been causatively‐linked to numerous pathophysiologic processes. The 48 kDa CN fragment (DCN) appears at high levels in human brain at early stages of cognitive decline associated with Alzheimer’s disease. DCN tends to show‐up in regions of frank amyloid and cerebrovascular pathology, especially in select subsets of astrocytes, both in humans and in animal models. Our goal was to develop monoclonal antibodies to DCN for use in neuropathology research.MethodA peptide encompassing the calpain sensitive region of the CN carboxyl terminus was used for antibody generation. Antibodies were screened in ELISAs against the immunizing peptide, but decision‐making screens were carried out as a Western analysis of 5XFAD mouse brain extracts, which express high levels of the DCN fragment.ResultWe identified 2 monoclonals, one of which (17E1) was highly reactive to DCN in Western blots, but not in ELISAs, of 5xFAD brain extracts. In contrast, the second antibody (26A6) reacted well against the peptide in ELISAs, but worked only modestly in Westerns. Importantly, neither antibody reacted with full‐length (60 kDa) CN, nor did antibodies detect DCN in extracts from healthy WT mice. In immunostaining assays, both antibodies strongly labeled subsets of GFAP‐positive astrocytes in 5xFAD mice with a high signal‐to‐background. Some GFAP‐negative cells also labeled positively for DCN. Consistent with Westerns, there was no antibody reactivity to WT mouse brain sections.ConclusionNew monoclonals are highly selective for the 48 kDa CN proteolytic fragment and label subsets of astrocytes, and possibly other cell types, under pathological conditions. These antibodies could be a useful tool for marking insidious brain pathology and identifying novel astrocyte phenotypes.

Ganglioside GD3 regulates dendritic growth in newborn neurons in adult mouse hippocampus via modulation of mitochondrial dynamics.

Ganglioside GD3, a major ganglioside species in neural stem cells, plays a crucial role in maintenance of the self-renewal capacity of these cells. However, its bioactivity in postnatally differentiated neurons in the neurogenic regions of adult brains has not been elucidated. Here, we describe for the first time that deletion of GD3 not only impairs neurotrophin-induced stem cell proliferation, but also alters the dendritic structure as well as the number of synapses of nascent neurons in the dentate gyrus of adult brain. When examining the behavioral phenotypes, GD3 synthase-knockout (GD3S-KO) mice displayed impairment in hippocampus-dependent memory function. To further gain insight into its cellular function, we examined GD3-binding partners from mouse brain extract using a GD3-specific monoclonal antibody, R24, followed by LC-MS/MS analysis and identified a mitochondrial fission protein, the dynamin-related protein-1 (Drp1), as a novel GD3-binding protein. Biochemical and imaging analyses revealed mitochondrial fragmentation in GD3-depleted dentate gyrus neurons, suggesting that GD3 is essential for the mitochondrial Drp1 turnover that is required for efficient mitochondrial fission. These results suggest that GD3 is required for proper dendritic and spine maturation of newborn neurons in adult brain through the regulation of mitochondrial dynamics.

Effect of (m)VD-hemopressin against Aβ1-42-induced oxidative stress and apoptosis in mouse hippocampal neurons

Alzheimer’s disease (AD) is a serious neurodegenerative disease. Senile plaques (SPs) composed of amyloid-β (Aβ) are typical features of AD. Aβ plays a key role in the disease and has the ability to induce other pathological characteristics of AD, including oxidative stress injury. (m)VD-hemopressin (VD), a peptide derived from mouse brain extracts, can bind cannabinoid 1 receptor (CB1R) as an agonist. Our previous report indicated that VD reverses memory impairment induced by Aβ1-42 in mice. This study aimed to clarify the mechanism by which VD protects hippocampal neurons against Aβ1-42-induced impairment. Our results showed that VD inhibited oxidative stress injury induced by Aβ1-42, as demonstrated by the VD-induced reversal of the upregulation of reactive oxygen species (ROS) and the intracellular lipid peroxidation product malondialdehyde (MDA) and the downregulation of the activities of the antioxidative enzymes catalase (CAT) and glutathione peroxidase (GSH-PX) in mouse hippocampal neurons. We also found that VD restored the decrease in cell growth and viability induced by Aβ1-42 and reversed Aβ1-42-induced apoptosis mediated by the apoptosis-associated proteins Bcl-2 and Bax. However, cotreatment with AM251 (an antagonist of CB1R) blocked the effects of VD. In brief, this study suggested that through CB1R, VD reversed the impairment of cell growth and viability, oxidative stress injury and apoptosis induced by Aβ1-42. Therefore, VD may be a promising agent for the treatment of diseases that involve oxidative stress injury and apoptosis induced by Aβ1-42, such as AD.

Different Amyloid-β Self-Assemblies Have Distinct Effects on Intracellular Tau Aggregation.

Alzheimer’s disease (AD) pathology is characterized by the aggregation of beta-amyloid (Aβ) and tau in the form of amyloid plaques and neurofibrillary tangles in the brain. It has been found that a synergistic relationship between these two proteins may contribute to their roles in disease progression. However, how Aβ and tau interact has not been fully characterized. Here, we analyze how tau seeding or aggregation is influenced by different Aβ self-assemblies (fibrils and oligomers). Our cellular assays utilizing tau biosensor cells show that transduction of Aβ oligomers into the cells greatly enhances seeded tau aggregation in a concentration-dependent manner. In contrast, transduced Aβ fibrils slightly reduce tau seeding while untransduced Aβ fibrils promote it. We also observe that the transduction of α-synuclein fibrils, another amyloid protein, has no effect on tau seeding. The enhancement of tau seeding by Aβ oligomers was confirmed using tau fibril seeds derived from both recombinant tau and PS19 mouse brain extracts containing human tau. Our findings highlight the importance of considering the specific form and cellular location of Aβ self-assembly when studying the relationship between Aβ and tau in future AD therapeutic development.