Apolipoprotein E4 Impairs Neuronal Insulin Signaling by Trapping Insulin Receptor in the Endosomes.

Diabetes and impaired brain insulin signaling are linked to the pathogenesis of Alzheimer's disease (AD). The association between diabetes and AD-associated amyloid pathology is stronger among carriers of the apolipoprotein E (APOE) ε4 gene allele, the strongest genetic risk factor for late-onset AD. Here we report that apoE4 impairs neuronal insulin signaling in human apoE-targeted replacement (TR) mice in an age-dependent manner. High-fat diet (HFD) accelerates these effects in apoE4-TR mice at middle age. In primary neurons, apoE4 interacts with insulin receptor and impairs its trafficking by trapping it in the endosomes, leading to impaired insulin signaling and insulin-stimulated mitochondrial respiration and glycolysis. In aging brains, the increased apoE4 aggregation and compromised endosomal function further exacerbate the inhibitory effects of apoE4 on insulin signaling and related functions. Together, our study provides novel mechanistic insights into the pathogenic mechanisms of apoE4 and insulin resistance in AD.

Antibody Binding Specificity for Kappa (Vκ) Light Chain-containing Human (IgM) Antibodies: Polysialic Acid (PSA) Attached to NCAM as a Case Study.

Antibodies of the IgM isotype are often neglected as potential therapeutics in human trials, animal models of human diseases as well as detecting agents in standard laboratory techniques. In contrast, several human IgMs demonstrated proof of efficacy in cancer models and models of CNS disorders including multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS). Reasons for their lack of consideration include difficulties to express, purify and stabilize IgM antibodies, challenge to identify (non-protein) antigens, low affinity binding and fundamental knowledge gaps in carbohydrate and lipid research. This manuscript uses HIgM12 as an example to provide a detailed protocol to detect antigens by Western blotting, immunoprecipitations and immunocytochemistry. HIgM12 targets polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM). Early postnatal mouse brain tissue from wild type (WT) and NCAM knockout (KO) mice lacking the three major central nervous system (CNS) splice variants NCAM180, 140 and 120 was used to evaluate the importance of NCAM for binding to HIgM12. Further enzymatic digestion of CNS tissue and cultured CNS cells using endoneuraminidases led us to identify PSA as the specific binding epitope for HIgM12.

Apolipoprotein E as a Therapeutic Target in Alzheimer's Disease: A Review of Basic Research and Clinical Evidence.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder that causes progressive cognitive decline. The majority of AD cases are sporadic and late-onset (>65 years old) making it the leading cause of dementia in the elderly. While both genetic and environmental factors contribute to the development of late-onset AD (LOAD), APOE polymorphism is a major genetic risk determinant for LOAD. In humans, the APOE gene has three major allelic variants: ε2, ε3, and ε4, of which APOE ε4 is the strongest genetic risk factor for LOAD, whereas APOE ε2 is protective. Mounting evidence suggests that APOE ε4 contributes to AD pathogenesis through multiple pathways including facilitated amyloid-ß deposition, increased tangle formation, synaptic dysfunction, exacerbated neuroinflammation, and cerebrovascular defects. Since APOE modulates multiple biological processes through its corresponding protein apolipoprotein E (apoE), APOE gene and apoE properties have been a promising target for therapy and drug development against AD. In this review, we summarize the current evidence regarding how the APOE ε4 allele contributes to the pathogenesis of AD and how relevant therapeutic approaches can be developed to target apoE-mediated pathways in AD.

Impact of sex and APOE4 on cerebral amyloid angiopathy in Alzheimer's disease.

Cerebral amyloid angiopathy (CAA) often coexists with Alzheimer's disease (AD). APOE4 is a strong genetic risk factor for both AD and CAA. Sex-dependent differences have been shown in AD as well as in cerebrovascular diseases. Therefore, we examined the effects of APOE4, sex, and pathological components on CAA in AD subjects. A total of 428 autopsied brain samples from pathologically confirmed AD cases were analyzed. CAA severity was histologically scored in inferior parietal, middle frontal, motor, superior temporal and visual cortexes. In addition, subgroups with severe CAA (n = 60) or without CAA (n = 39) were subjected to biochemical analysis of amyloid-ß (Aß) and apolipoprotein E (apoE) by ELISA in the temporal cortex. After adjusting for age, Braak neurofibrillary tangle stage and Thal amyloid phase, we found that overall CAA scores were higher in males than females. Furthermore, carrying one or more APOE4 alleles was associated with higher overall CAA scores. Biochemical analysis revealed that the levels of detergent-soluble and detergent-insoluble Aß40, and insoluble apoE were significantly elevated in individuals with severe CAA or APOE4. The ratio of Aß40/Aß42 in insoluble fractions was also increased in the presence of CAA or APOE4, although it was negatively associated with male sex. Levels of insoluble Aß40 were positively associated with those of insoluble apoE, which were strongly influenced by CAA status. Pertaining to insoluble Aß42, the levels of apoE correlated regardless of CAA status. Our results indicate that sex and APOE genotypes differentially influence the presence and severity of CAA in AD, likely by affecting interaction and aggregation of Aß40 and apoE.

Antiviral Protection via RdRP-Mediated Stable Activation of Innate Immunity.

For many emerging and re-emerging infectious diseases, definitive solutions via sterilizing adaptive immunity may require years or decades to develop, if they are even possible. The innate immune system offers alternative mechanisms that do not require antigen-specific recognition or a priori knowledge of the causative agent. However, it is unclear whether effective stable innate immune system activation can be achieved without triggering harmful autoimmunity or other chronic inflammatory sequelae. Here, we show that transgenic expression of a picornavirus RNA-dependent RNA polymerase (RdRP), in the absence of other viral proteins, can profoundly reconfigure mammalian innate antiviral immunity by exposing the normally membrane-sequestered RdRP activity to sustained innate immune detection. RdRP-transgenic mice have life-long, quantitatively dramatic upregulation of 80 interferon-stimulated genes (ISGs) and show profound resistance to normally lethal viral challenge. Multiple crosses with defined knockout mice (Rag1, Mda5, Mavs, Ifnar1, Ifngr1, and Tlr3) established that the mechanism operates via MDA5 and MAVS and is fully independent of the adaptive immune system. Human cell models recapitulated the key features with striking fidelity, with the RdRP inducing an analogous ISG network and a strict block to HIV-1 infection. This RdRP-mediated antiviral mechanism does not depend on secondary structure within the RdRP mRNA but operates at the protein level and requires RdRP catalysis. Importantly, despite lifelong massive ISG elevations, RdRP mice are entirely healthy, with normal longevity. Our data reveal that a powerfully augmented MDA5-mediated activation state can be a well-tolerated mammalian innate immune system configuration. These results provide a foundation for augmenting innate immunity to achieve broad-spectrum antiviral protection.

Apolipoprotein E Is a Ligand for Triggering Receptor Expressed on Myeloid Cells 2 (TREM2).

Several heterozygous missense mutations in the triggering receptor expressed on myeloid cells 2 (TREM2) have recently been linked to risk for a number of neurological disorders including Alzheimer disease (AD), Parkinson disease, and frontotemporal dementia. These discoveries have re-ignited interest in the role of neuroinflammation in the pathogenesis of neurodegenerative diseases. TREM2 is highly expressed in microglia, the resident immune cells of the central nervous system. Along with its adaptor protein, DAP12, TREM2 regulates inflammatory cytokine release and phagocytosis of apoptotic neurons. Here, we report apolipoprotein E (apoE) as a novel ligand for TREM2. Using a biochemical assay, we demonstrated high-affinity binding of apoE to human TREM2. The functional significance of this binding was highlighted by increased phagocytosis of apoE-bound apoptotic N2a cells by primary microglia in a manner that depends on TREM2 expression. Moreover, when the AD-associated TREM2-R47H mutant was used in biochemical assays, apoE binding was vastly reduced. Our data demonstrate that apoE-TREM2 interaction in microglia plays critical roles in modulating phagocytosis of apoE-bound apoptotic neurons and establish a critical link between two proteins whose genes are strongly linked to the risk for AD.

TREM2 in CNS homeostasis and neurodegenerative disease.

Myeloid-lineage cells accomplish a myriad of homeostatic tasks including the recognition of pathogens, regulation of the inflammatory milieu, and mediation of tissue repair and regeneration. The innate immune receptor and its adaptor protein­triggering receptor expressed on myeloid cells 2 (TREM2) and DNAX-activating protein of 12 kDa (DAP12)­possess the ability to modulate critical cellular functions via crosstalk with diverse signaling pathways. As such, mutations in TREM2 and DAP12 have been found to be associated with a range of disease phenotypes. In particular, mutations in TREM2 increase the risk for Alzheimer's disease and other neurodegenerative disorders. The leading hypothesis is that microglia, the resident immune cells of the central nervous system, are the major myeloid cells affected by dysregulated TREM2-DAP12 function. Here, we review how impaired signaling by the TREM2-DAP12 pathway leads to altered immune responses in phagocytosis, cytokine production, and microglial proliferation and survival, thus contributing to disease pathogenesis.

Abbreviated exposure to hypoxia is sufficient to induce CNS dysmyelination, modulate spinal motor neuron composition, and impair motor development in neonatal mice.

Neonatal white matter injury (nWMI) is an increasingly common cause of cerebral palsy that results predominantly from hypoxic injury to progenitor cells including those of the oligodendrocyte lineage. Existing mouse models of nWMI utilize prolonged periods of hypoxia during the neonatal period, require complex cross-fostering and exhibit poor growth and high mortality rates. Abnormal CNS myelin composition serves as the major explanation for persistent neuro-motor deficits. Here we developed a simplified model of nWMI with low mortality rates and improved growth without cross-fostering. Neonatal mice are exposed to low oxygen from postnatal day (P) 3 to P7, which roughly corresponds to the period of human brain development between gestational weeks 32 and 36. CNS hypomyelination is detectable for 2-3 weeks post injury and strongly correlates with levels of body and brain weight loss. Immediately following hypoxia treatment, cell death was evident in multiple brain regions, most notably in superficial and deep cortical layers as well as the subventricular zone progenitor compartment. PDGFαR, Nkx2.2, and Olig2 positive oligodendrocyte progenitor cell were significantly reduced until postnatal day 27. In addition to CNS dysmyelination we identified a novel pathological marker for adult hypoxic animals that strongly correlates with life-long neuro-motor deficits. Mice reared under hypoxia reveal an abnormal spinal neuron composition with increased small and medium diameter axons and decreased large diameter axons in thoracic lateral and anterior funiculi. Differences were particularly pronounced in white matter motor tracts left and right of the anterior median fissure. Our findings suggest that 4 days of exposure to hypoxia are sufficient to induce experimental nWMI in CD1 mice, thus providing a model to test new therapeutics. Pathological hallmarks of this model include early cell death, decreased OPCs and hypomyelination in early postnatal life, followed by dysmyelination, abnormal spinal neuron composition, and neuro-motor deficits in adulthood.

Polysialic acid as an antigen for monoclonal antibody HIgM12 to treat multiple sclerosis and other neurodegenerative disorders.

CNS regeneration is a desirable goal for diseases of brain and spinal cord. Current therapeutic strategies for the treatment of multiple sclerosis (MS) aim to eliminate detrimental effects of the immune system, so far without reversing disability or affecting long-term prognosis in patients. Approachable molecular targets that stimulate CNS repair are not part of the clinical praxis or have not been identified yet. The purpose of this study was to identify the molecular target of the human monoclonal antibody HIgM12. HIgM12 reverses motor deficits in chronically demyelinated mice, a model of MS. Here, we identified polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM) as the antigen for HIgM12 by using different NCAM knockout strains and through PSA removal from the NCAM protein core. Antibody binding to CNS tissue and primary cells, antibody-mediated cell adhesion, and neurite outgrowth on HIgM12-coated nitrocellulose was detected only in the presence of PSA as assessed by western blotting, immunoprecipitation, immunocytochemistry, and histochemistry. We conclude that HIgM12 mediates its in vivo and in vitro effects through binding to PSA and has the potential to be an effective therapy for MS and neurodegenerative diseases. The human antibody HIgM12 stimulates neurite outgrowth in vitro and promotes function in chronically demyelinated mice, a model of multiple sclerosis. The cellular antigen for HIgM12 was undetermined. Here, we identified polysialic acid attached to NCAM (neural cell adhesion molecule) as the cellular target for HIgM12. This includes glial fibrillary acidic protein (GFAP)-positive mouse astrocytes (GFAP, red; HIgM12, green; DAPI, blue) among other cell types of the central nervous system. These findings indicate a new strategy for the treatment of neuro-motor disorders including multiple sclerosis.

A human anti-polysialic acid antibody as a potential treatment to improve function in multiple sclerosis patients.

We previously identified a human monoclonal antibody, termed HIgM12 that stimulates spontaneous locomotor activity in a chronically demyelinating mouse model of multiple sclerosis. When tested as a molecular substrate, HIgM12 stimulated neurite outgrowth in vitro. We recently reported that polysialic acid (PSA) attached to the neural cell adhesion molecule (NCAM) is one of the cellular antigens for HIgM12. Fluorescent double-labeling of astrocytes using HIgM12 and commercially available anti-PSA antibody showed dramatic co-localization. Neural tissue homogenates and primary CNS cultures from mice lacking the three major NCAM splice variants NCAM180, NCAM140 and NCAM120 (NCAM KO) were no longer able to bind HIgM12. Furthermore, enzymatic digestion of PSA on wild type (WT) glia abolished HIgM12-binding. Moreover, neurons and glia from NCAM KO animals did not attach to HIgM12-coated nitrocellulose in neurite outgrowth assays. We conclude that HIgM12 targets PSA attached to NCAM, and that the PSA moiety mediates neuronal and glial adhesion and subsequent neurite outgrowth in our in vitro assay. Therefore, this anti-PSA antibody may serve as a future therapeutic to stimulate functional improvement in multiple sclerosis patients and other neurodegenerative diseases.

Cellular targets and mechanistic strategies of remyelination-promoting IgMs as part of the naturally occurring autoantibody repertoire.

Immunoglobulins with germline sequences occur in invertebrates and vertebrates and are named naturally occurring autoantibodies (NAbs). NAbs may target foreign antigens, self- or altered self-components and are part of the normal immunoglobulin repertoire. Accumulating evidence indicates that naturally occurring antibodies can act as systemic surveillance molecules, which tag, damaged or stressed cells, invading pathogens and toxic cellular debris for elimination by the immune system. In addition to acting as detecting molecules, certain types of NAbs actively signal in different cell types with a broad range of responses from induction of apoptosis in cancer cells to stimulation of remyelination in glial cells. This review emphasizes functions and characteristics of NAbs with focus on remyelination-promoting mouse and human antibodies. Human remyelination-promoting NAbs are potential therapeutics to combat a wide spectrum of disease processes including demyelinating diseases like multiple sclerosis. We will highlight the identified glycosphingolipid (SL) antigens of polyreactive remyelination-promoting antibodies and their proposed mechanism(s) of action. The nature of the identified antigens suggests a lipid raft-based mechanism for remyelination-promoting antibodies with SLs as most essential raft components. However, accumulating evidence also suggests involvement of other antigens in stimulation of remyelination, which will be discussed in the text.

Selective uptake and biological consequences of environmentally relevant antidepressant pharmaceutical exposures on male fathead minnows.

Antidepressant pharmaceuticals have been reported in wastewater effluent at the nanogram to low microgram-per-liter range, and include bupropion (BUP), fluoxetine (FLX), sertraline (SER), and venlafaxine (VEN). To assess the effects of antidepressants on reproductive anatomy, physiology, and behavior, adult male fathead minnows (Pimephales promelas) were exposed for 21 days either to a single concentration of the antidepressants FLX, SER, VEN, or BUP, or to an antidepressant mixture. The data demonstrated that exposure to VEN (305 ng/L and 1104 ng/L) and SER (5.2 ng/L) resulted in mortality. Anatomical alterations were noted within the testes of fish exposed to SER and FLX, both modulators of the neurotransmitter serotonin. Additionally, FLX at 28 ng/L induced vitellogenin in male fish--a common endpoint for estrogenic endocrine disruption. Significant alterations in male secondary sex characteristics were noted with single exposures. Effects of single compound exposures neither carried over, nor became additive in the antidepressant mixtures, and reproductive behavior was not affected. Analysis of brain tissues from the exposed fish suggested increased uptake of FLX, SER and BUP and minimal uptake of VEN when compared to exposure water concentrations. Furthermore, the only metabolite detected consistently in the brain tissues was norfluoxetine. Similar trends of uptake by brain tissue were observed when fish were exposed to antidepressant mixtures. The present study demonstrates that anatomy and physiology, but not reproductive behavior, can be disrupted by exposure to environmental concentrations of some antidepressants. The observation that antidepressant uptake into fish tissues is selective may have consequences on assessing the mode-of-action and effects of these compounds in future studies.

Antidepressants at environmentally relevant concentrations affect predator avoidance behavior of larval fathead minnows (Pimephales promelas).

The effects of embryonic and larval exposure to environmentally relevant (ng/L) concentrations of common antidepressants, fluoxetine, sertraline, venlafaxine, and bupropion (singularly and in mixture) on C-start escape behavior were evaluated in fathead minnows (Pimephales promelas). Embryos (postfertilization until hatching) were exposed for 5 d and, after hatching, were allowed to grow in control well water until 12 d old. Similarly, posthatch fathead minnows were exposed for 12 d to these compounds. High-speed (1,000 frames/s) video recordings of escape behavior were collected and transferred to National Institutes of Health Image for frame-by-frame analysis of latency periods, escape velocities, and total escape response (combination of latency period and escape velocity). When tested 12 d posthatch, fluoxetine and venlafaxine adversely affected C-start performance of larvae exposed as embryos. Conversely, larvae exposed for 12 d posthatch did not exhibit altered escape responses when exposed to fluoxetine but were affected by venlafaxine and bupropion exposure. Mixtures of these four antidepressant pharmaceuticals slowed predator avoidance behaviors in larval fathead minnows regardless of the exposure window. The direct impact of reduced C-start performance on survival and, ultimately, reproductive fitness provides an avenue to assess the ecological relevance of exposure in an assay of relatively short duration.