ApoE4 markedly exacerbates tau-mediated neurodegeneration in a mouse model of tauopathy.

APOE4 is the strongest genetic risk factor for late-onset Alzheimer disease. ApoE4 increases brain amyloid-ß pathology relative to other ApoE isoforms. However, whether APOE independently influences tau pathology, the other major proteinopathy of Alzheimer disease and other tauopathies, or tau-mediated neurodegeneration, is not clear. By generating P301S tau transgenic mice on either a human ApoE knock-in (KI) or ApoE knockout (KO) background, here we show that P301S/E4 mice have significantly higher tau levels in the brain and a greater extent of somatodendritic tau redistribution by three months of age compared with P301S/E2, P301S/E3, and P301S/EKO mice. By nine months of age, P301S mice with different ApoE genotypes display distinct phosphorylated tau protein (p-tau) staining patterns. P301S/E4 mice develop markedly more brain atrophy and neuroinflammation than P301S/E2 and P301S/E3 mice, whereas P301S/EKO mice are largely protected from these changes. In vitro, E4-expressing microglia exhibit higher innate immune reactivity after lipopolysaccharide treatment. Co-culturing P301S tau-expressing neurons with E4-expressing mixed glia results in a significantly higher level of tumour-necrosis factor-α (TNF-α) secretion and markedly reduced neuronal viability compared with neuron/E2 and neuron/E3 co-cultures. Neurons co-cultured with EKO glia showed the greatest viability with the lowest level of secreted TNF-α. Treatment of P301S neurons with recombinant ApoE (E2, E3, E4) also leads to some neuronal damage and death compared with the absence of ApoE, with ApoE4 exacerbating the effect. In individuals with a sporadic primary tauopathy, the presence of an ε4 allele is associated with more severe regional neurodegeneration. In individuals who are positive for amyloid-ß pathology with symptomatic Alzheimer disease who usually have tau pathology, ε4-carriers demonstrate greater rates of disease progression. Our results demonstrate that ApoE affects tau pathogenesis, neuroinflammation, and tau-mediated neurodegeneration independently of amyloid-ß pathology. ApoE4 exerts a 'toxic' gain of function whereas the absence of ApoE is protective.

Amyloid-ß and Tau at the Crossroads of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common form of dementia characterized neuropathologically by senile plaques and neurofibrillary tangles (NFTs). Early breakthroughs in AD research led to the discovery of amyloid-ß as the major component of senile plaques and tau protein as the major component of NFTs. Shortly following the identification of the amyloid-ß (Aß) peptide was the discovery that a genetic mutation in the amyloid precursor protein (APP), a type1 transmembrane protein, can be a cause of autosomal dominant familial AD (fAD). These discoveries, coupled with other breakthroughs in cell biology and human genetics, have led to a theory known as the "amyloid hypothesis", which postulates that amyloid-ß is the predominant driving factor in AD development. Nonetheless, more recent advances in imaging analysis, biomarkers and mouse models are now redefining this original hypothesis, as it is likely amyloid-ß, tau and other pathophysiological mechanism such as inflammation, come together at a crossroads that ultimately leads to the development of AD.

Ubiquitin-Synaptobrevin Fusion Protein Causes Degeneration of Presynaptic Motor Terminals in Mice.

Protein aggregates containing ubiquitin (Ub) are commonly observed in neurodegenerative disorders, implicating the involvement of the ubiquitin proteasome system (UPS) in their pathogenesis. Here, we aimed to generate a mouse model for monitoring UPS function using a green fluorescent protein (GFP)-based substrate that carries a "noncleavable" N-terminal ubiquitin moiety (Ub(G76V)). We engineered transgenic mice expressing a fusion protein, consisting of the following: (1) Ub(G76V), GFP, and a synaptic vesicle protein synaptobrevin-2 (Ub(G76V)-GFP-Syb2); (2) GFP-Syb2; or (3) Ub(G76V)-GFP-Syntaxin1, all under the control of a neuron-specific Thy-1 promoter. As expected, Ub(G76V)-GFP-Syb2, GFP-Syb2, and Ub(G76V)-GFP-Sytaxin1 were highly expressed in neurons, such as motoneurons and motor nerve terminals of the neuromuscular junction (NMJ). Surprisingly, Ub(G76V)-GFP-Syb2 mice developed progressive adult-onset degeneration of motor nerve terminals, whereas GFP-Syb2 and Ub(G76V)-GFP-Syntaxin1 mice were normal. The degeneration of nerve terminals in Ub(G76V)-GFP-Syb2 mice was preceded by a progressive impairment of synaptic transmission at the NMJs. Biochemical analyses demonstrated that Ub(G76V)-GFP-Syb2 interacted with SNAP-25 and Syntaxin1, the SNARE partners of synaptobrevin. Ultrastructural analyses revealed a marked reduction in synaptic vesicle density, accompanying an accumulation of tubulovesicular structures at presynaptic nerve terminals. These morphological defects were largely restricted to motor nerve terminals, as the ultrastructure of motoneuron somata appeared to be normal at the stages when synaptic nerve terminals degenerated. Furthermore, synaptic vesicle endocytosis and membrane trafficking were impaired in Ub(G76V)-GFP-Syb2 mice. These findings indicate that Ub(G76V)-GFP-Syb2 may compete with endogenous synaptobrevin, acting as a gain-of-function mutation that impedes SNARE function, resulting in the depletion of synaptic vesicles and degeneration of the nerve terminals. SIGNIFICANCE STATEMENT: Degeneration of motor nerve terminals occurs in amyotrophic lateral sclerosis (ALS) patients as well as in mouse models of ALS, leading to progressive paralysis. What causes a motor nerve terminal to degenerate remains unknown. Here we report on transgenic mice expressing a ubiquitinated synaptic vesicle protein (Ub(G76V)-GFP-Syb2) that develop progressive degeneration of motor nerve terminals. These mice may serve as a model for further elucidating the underlying cellular and molecular mechanisms of presynaptic nerve terminal degeneration.

An OBSL1-Cul7Fbxw8 ubiquitin ligase signaling mechanism regulates Golgi morphology and dendrite patterning.

The elaboration of dendrites in neurons requires secretory trafficking through the Golgi apparatus, but the mechanisms that govern Golgi function in neuronal morphogenesis in the brain have remained largely unexplored. Here, we report that the E3 ubiquitin ligase Cul7(Fbxw8) localizes to the Golgi complex in mammalian brain neurons. Inhibition of Cul7(Fbxw8) by independent approaches including Fbxw8 knockdown reveals that Cul7(Fbxw8) is selectively required for the growth and elaboration of dendrites but not axons in primary neurons and in the developing rat cerebellum in vivo. Inhibition of Cul7(Fbxw8) also dramatically impairs the morphology of the Golgi complex, leading to deficient secretory trafficking in neurons. Using an immunoprecipitation/mass spectrometry screening approach, we also uncover the cytoskeletal adaptor protein OBSL1 as a critical regulator of Cul7(Fbxw8) in Golgi morphogenesis and dendrite elaboration. OBSL1 forms a physical complex with the scaffold protein Cul7 and thereby localizes Cul7 at the Golgi apparatus. Accordingly, OBSL1 is required for the morphogenesis of the Golgi apparatus and the elaboration of dendrites. Finally, we identify the Golgi protein Grasp65 as a novel and physiologically relevant substrate of Cul7(Fbxw8) in the control of Golgi and dendrite morphogenesis in neurons. Collectively, these findings define a novel OBSL1-regulated Cul7(Fbxw8) ubiquitin signaling mechanism that orchestrates the morphogenesis of the Golgi apparatus and patterning of dendrites, with fundamental implications for our understanding of brain development.

A molecular pathway of neurodegeneration linking alpha-synuclein to ApoE and Abeta peptides.

Pathogenic aggregates of alpha-synuclein are thought to contribute to the development of Parkinson's disease. Inclusion bodies containing alpha-synuclein are present in Parkinson's disease and other neurodegenerative diseases, including Alzheimer's disease. Moreover, alpha-synuclein mutations are found in cases of familial Parkinson's disease, and transgenic overexpression of alpha-synuclein causes neurodegeneration in mice. The molecular mechanisms involved, however, remain incompletely understood. Here we show that, in transgenic mice, alpha-synuclein induced neurodegeneration involves activation of the ubiquitin/proteasome system, a massive increase in apolipoprotein E (ApoE) levels and accumulation of insoluble mouse Abeta. ApoE was not protective, but was injurious, as deletion of ApoE delayed the neurodegeneration caused by alpha-synuclein and suppressed the accumulation of Abeta. Our data reveal a molecular link between central pathogenic mechanisms implicated in Parkinson's disease and Alzheimer's disease and suggest that intracellular alpha-synuclein is pathogenic, at least in part, by activation of extracellular signaling pathways involving ApoE.

Astrocytic α2-Na+/K+ ATPase inhibition suppresses astrocyte reactivity and reduces neurodegeneration in a tauopathy mouse model.

Alzheimer's disease (AD) is the most dominant form of dementia characterized by the deposition of extracellular amyloid plaques and intracellular neurofibrillary tau tangles (NFTs). In addition to these pathologies, an emerging pathophysiological mechanism that influences AD is neuroinflammation. Astrocytes are a vital type of glial cell that contribute to neuroinflammation, and reactive astrocytes, or astrogliosis, are a well-known pathological feature of AD. However, the mechanisms by which astrocytes contribute to the neurodegenerative process in AD have not been fully elucidated. Here, we showed that astrocytic α2-Na+/K+ adenosine triphosphatase (α2-NKA) is elevated in postmortem human brain tissue from AD and progressive nuclear palsy, a primary tauopathy. The increased astrocytic α2-NKA was also recapitulated in a mouse model of tauopathy. Pharmacological inhibition of α2-NKA robustly suppressed neuroinflammation and reduced brain atrophy. In addition, α2-NKA knockdown in tauopathy mice halted the accumulation of tau pathology. We also demonstrated that α2-NKA promoted tauopathy, in part, by regulating the proinflammatory protein lipocalin-2 (Lcn2). Overexpression of Lcn2 in tauopathy mice increased tau pathology, and prolonged Lcn2 exposure to primary neurons promoted tau uptake in vitro. These studies collectively highlight the contribution of reactive astrocytes to tau pathogenesis in mice and define α2-NKA as a major regulator of astrocytic-dependent neuroinflammation.

Targeting tauopathy with engineered tau-degrading intrabodies.

BACKGROUND: The accumulation of pathological tau is the main component of neurofibrillary tangles and other tau aggregates in several neurodegenerative diseases, referred to as tauopathies. Recently, immunotherapeutic approaches targeting tau have been demonstrated to be beneficial in decreasing tauopathy in animal models. We previously found that passive immunotherapy with anti-tau antibody to human tau or expression of an anti-tau secreted single-chain variable fragment (scFv) in the central nervous system of a mouse model of tauopathy decreased but did not remove all tau-associated pathology. Although these and other studies demonstrate that conventional immunotherapeutic approaches targeting tau can influence tau pathogenesis, the majority of pathological tau remains in the cytosol of cells, not typically accessible to an extracellular antibody. Therefore, we reasoned targeting intracellular tau might be more efficacious in preventing or decreasing tauopathy. METHODS: By utilizing our anti-tau scFv, we generated anti-tau intrabodies for the expression in the cytosol of neurons. To enhance the degradation capacity of conventional intrabodies, we engineered chimeric anti-tau intrabodies fused to ubiquitin harboring distinct mutations that shuttle intracellular tau for either the proteasome or lysosomal mediated degradation. To evaluate the efficacy in delaying or eliminating tauopathy, we expressed our tau degrading intrabodies or controls in human tau transgenic mice by adeno-associated virus prior to overt tau pathology and after tau deposition. RESULTS: Our results demonstrate, the expression of chimeric anti-tau intrabodies significantly reduce tau protein levels in primary neuronal cultures expression human tau relative to a non-modified anti-tau intrabody. We found the expression of engineered tau-degrading intrabodies destined for proteasomal-mediated degradation are more effective in delaying or eliminating tauopathy than a conventional intrabody in aged human tau transgenic mice. CONCLUSION: This study, harnesses the strength of intrabodies that are amendable for targeting specific domains or modifications with the cell-intrinsic mechanisms that regulate protein degradation providing a new immunotherapeutic approach with potentially improved efficacy.

Targeting of nonlipidated, aggregated apoE with antibodies inhibits amyloid accumulation.

The apolipoprotein E E4 allele of the APOE gene is the strongest genetic factor for late-onset Alzheimer disease (LOAD). There is compelling evidence that apoE influences Alzheimer disease (AD) in large part by affecting amyloid ß (Aß) aggregation and clearance; however, the molecular mechanism underlying these findings remains largely unknown. Herein, we tested whether anti-human apoE antibodies can decrease Aß pathology in mice producing both human Aß and apoE4, and investigated the mechanism underlying these effects. We utilized APPPS1-21 mice crossed to apoE4-knockin mice expressing human apoE4 (APPPS1-21/APOE4). We discovered an anti-human apoE antibody, anti-human apoE 4 (HAE-4), that specifically recognizes human apoE4 and apoE3 and preferentially binds nonlipidated, aggregated apoE over the lipidated apoE found in circulation. HAE-4 also binds to apoE in amyloid plaques in unfixed brain sections and in living APPPS1-21/APOE4 mice. When delivered centrally or by peripheral injection, HAE-4 reduced Aß deposition in APPPS1-21/APOE4 mice. Using adeno-associated virus to express 2 different full-length anti-apoE antibodies in the brain, we found that HAE antibodies decreased amyloid accumulation, which was dependent on Fcγ receptor function. These data support the hypothesis that a primary mechanism for apoE-mediated plaque formation may be a result of apoE aggregation, as preferentially targeting apoE aggregates with therapeutic antibodies reduces Aß pathology and may represent a selective approach to treat AD.

Antibody Therapeutics Targeting Aß and Tau.

The astonishing findings that active and passive immunization against amyloid-ß (Aß) in mouse models of Alzheimer's disease (AD) dramatically decreased amyloid burden led to a rapid initiation of human clinical trials with much enthusiasm. However, methodological issues and adverse effects relating to these clinical trials arose, challenging the effectiveness and safety of these reagents. Efforts are now underway to develop safer immunotherapeutic approaches toward Aß and the treatment of individuals at risk for AD before or in the earliest stages of cognitive decline with new hopes. Furthermore, several studies have shown tau as a potential immunotherapeutic target for the treatment of tauopathy-related diseases including frontotemporal lobar dementia (FTLD). Both active and passive immunization targeting tau in mouse models of tauopathy effectively decreased tau pathology while improving cognitive performance. These preclinical studies have highlighted tau as an alternative target with much anticipation of clinical trials to be undertaken.

AAV-mediated expression of anti-tau scFvs decreases tau accumulation in a mouse model of tauopathy.

Tauopathies are characterized by the progressive accumulation of hyperphosphorylated, aggregated forms of tau. Our laboratory has previously demonstrated that passive immunization with an anti-tau antibody, HJ8.5, decreased accumulation of pathological tau in a human P301S tau-expressing transgenic (P301S-tg) mouse model of frontotemporal dementia/tauopathy. To investigate whether the Fc domain of HJ8.5 is required for the therapeutic effect, we engineered single-chain variable fragments (scFvs) derived from HJ8.5 with variable linker lengths, all specific to human tau. Based on different binding properties, we selected two anti-tau scFvs and tested their efficacy in vivo by adeno-associated virus-mediated gene transfer to the brain of P301S-tg mice. The scFvs significantly reduced levels of hyperphosphorylated, aggregated tau in brain tissue of P301S-tg mice, associated with a decrease in detergent-soluble tau species. Interestingly, these mice showed substantial levels of scFvs in the cerebrospinal fluid without significant effects on total extracellular tau levels. Therefore, our study provides a novel strategy for anti-tau immunotherapeutics that potentially limits a detrimental proinflammatory response.

Anti-tau antibody administration increases plasma tau in transgenic mice and patients with tauopathy.

Tauopathies are a group of disorders in which the cytosolic protein tau aggregates and accumulates in cells within the brain, resulting in neurodegeneration. A promising treatment being explored for tauopathies is passive immunization with anti-tau antibodies. We previously found that administration of an anti-tau antibody to human tau transgenic mice increased the concentration of plasma tau. We further explored the effects of administering an anti-tau antibody on plasma tau. After peripheral administration of an anti-tau antibody to human patients with tauopathy and to mice expressing human tau in the central nervous system, there was a dose-dependent increase in plasma tau. In mouse plasma, we found that tau had a short half-life of 8 min that increased to more than 3 hours after administration of anti-tau antibody. As tau transgenic mice accumulated insoluble tau in the brain, brain soluble and interstitial fluid tau decreased. Administration of anti-tau antibody to tau transgenic mice that had decreased brain soluble tau and interstitial fluid tau resulted in an increase in plasma tau, but this increase was less than that observed in tau transgenic mice without these brain changes. Tau transgenic mice subjected to acute neuronal injury using 3-nitropropionic acid showed increased interstitial fluid tau and plasma tau. These data suggest that peripheral administration of an anti-tau antibody results in increased plasma tau, which correlates with the concentration of extracellular and soluble tau in the brain.

An α2-Na/K ATPase/α-adducin complex in astrocytes triggers non-cell autonomous neurodegeneration.

Perturbations of astrocytes trigger neurodegeneration in several diseases, but the glial cell-intrinsic mechanisms that induce neurodegeneration remain poorly understood. We found that a protein complex of α2-Na/K ATPase and α-adducin was enriched in astrocytes expressing mutant superoxide dismutase 1 (SOD1), which causes familial amyotrophic lateral sclerosis (ALS). Knockdown of α2-Na/K ATPase or α-adducin in mutant SOD1 astrocytes protected motor neurons from degeneration, including in mutant SOD1 mice in vivo. Heterozygous disruption of the α2-Na/K ATPase gene suppressed degeneration in vivo and increased the lifespan of mutant SOD1 mice. The pharmacological agent digoxin, which inhibits Na/K ATPase activity, protected motor neurons from mutant SOD1 astrocyte-induced degeneration. Notably, α2-Na/K ATPase and α-adducin were upregulated in spinal cord of sporadic and familial ALS patients. Collectively, our findings define chronic activation of the α2-Na/K ATPase/α-adducin complex as a critical glial cell-intrinsic mechanism of non-cell autonomous neurodegeneration, with implications for potential therapies for neurodegenerative diseases.

The X-linked intellectual disability protein PHF6 associates with the PAF1 complex and regulates neuronal migration in the mammalian brain.

Intellectual disability is a prevalent disorder that remains incurable. Mutations of the X-linked protein PHF6 cause the intellectual disability disorder Börjeson-Forssman-Lehmann syndrome (BFLS). However, the biological role of PHF6 relevant to BFLS pathogenesis has remained unknown. We report that knockdown of PHF6 profoundly impairs neuronal migration in the mouse cerebral cortex in vivo, leading to the formation of white matter heterotopias displaying neuronal hyperexcitability. We find that PHF6 physically associates with the PAF1 transcription elongation complex, and inhibition of PAF1 phenocopies the PHF6 knockdown-induced migration phenotype in vivo. We also identify Neuroglycan C/Chondroitin sulfate proteoglycan 5 (NGC/CSPG5), a potential schizophrenia susceptibility gene, as a critical downstream target of PHF6 in the control of neuronal migration. These findings define PHF6, PAF1, and NGC/CSPG5 as components of a cell-intrinsic transcriptional pathway that orchestrates neuronal migration in the brain, with important implications for the pathogenesis of developmental disorders of cognition.

Helping Gulf shrimpers adopt safety measures: importance of partnerships and research to practice.

Commercial fishing continues to be a dangerous line of work. There are many hazards and the work is complex, even on a small scale. Along the United States Gulf Coast, the make-up of the commercial fishing population is diverse, with many Vietnamese shrimpers. Cultural barriers can interfere with critical communication and with receptivity to necessary safety training. In the course of studying these factors, it became apparent that language was a significant barrier among Vietnamese shrimp fishermen learning sound signals and making Mayday calls, potentially contributing to adverse events. This article is a qualitative description of a pilot project in response to this observation and aimed at the development of a model simulating the bridge of a commercial fishing vessel (including horn blast and radio). The model is used to improve knowledge and skills of the fishermen by providing instruction in Vietnamese. As a Mayday call must be made in English, instructional aids are provided to assist fishermen in the exercise. This example of research to practice (r2p) demonstrates how research findings may enhance acquisition of safety knowledge and skills through development of these types of models as well sustainable instructional tools like the multi-lingual interactive CD described here. It further illustrates the importance of partnerships in the design and delivery of workplace safety training interventions. The model, instructional aids, and CD are timely as they coincide with new regulation which mandates certification of these competencies or skills.

Factors influencing safety among a group of commercial fishermen along the Texas Gulf Coast.

The commercial fishing trades are among the most dangerous jobs in the world. Little published information exists regarding some populations of commercial fishermen such as along the United States Gulf Coast. Studying these unique and often vulnerable groups is important to characterize potential influences on or barriers to safety in anticipation of designing interventions that can change safety behaviors. Working closely with the United States Coast Guard (USCG), a cross-sectional convenience sample of Gulf Coast shrimp fishermen in and near the Port of Galveston, Texas, was surveyed. The survey included demographic factors and broadly covered areas such as type of work and fishing activities, general or global perceptions and beliefs related to safety and accidents, self-report of ability to use safety equipment or apply procedures aboard vessel, and training considerations. Surveys were obtained following informed consent (n = 133). Of the participants, 96.7% were male with 60.9% ≥40 years old. A majority were of Asian descent (57.1% of all fishermen, 82.1% of shrimp fishermen). Over half claimed to speak little or no English and nearly 60% considered the job to be very safe to neutral. A third to half of respondents expressed doubt about their knowledge of using essential safety equipment in the event of emergency. A large portion of the participants preferred hands-on safety training (40.6%). Important findings about this group of commercial fishermen will help with future development of effective prevention practices through the delivery of culturally appropriate safety awareness training. One element that must be addressed in training programs is to increase the awareness among fishermen about the severe occupational risks inherent in this type of work. Community trust and collaborative partnerships are essential to the success of such initiatives.

Cultural influences on safety and health education among Vietnamese fishermen.

Every ethnic group has its own cultural background and history that influences how it views health behaviors. By virtue of their work history, many Vietnamese have pursued the fishing industry when migrating to the United States. Even though the fishing trades are among the most dangerous jobs in the world, there has been little attention in the literature to the significant role that culture plays in the expression and experience of occupational health practices among Vietnamese shrimp fishermen. Three focus group sessions were conducted to identify factors that hinder or facilitate receptivity to available training and to guide culturally appropriate content. Participants were recruited using purposive sampling among various Vietnamese shrimp fishermen communities in Texas. Utilizing a series of open-ended questions, sessions were conducted in their native language among owners/captains/deckhands; support infrastructure--adult family members and religious/community leaders; and industry management and insurance representatives. Translations of transcribed documents were compared with simultaneous translations to ensure thematic consistency. Conducting hands-on training among Vietnamese by experienced fishermen, specifically targeting captains, was considered key to safety culture on the vessels. Findings of the study support that training should occur in a variety of formats (hands-on), but should be periodic, current, practical, convenient, taught in the primary language of the audience by an experienced individual, culminate in a certificate of completion, and target captains first. These findings illustrate the importance of considering cultural factors in the design of workplace interventions that focus on changes in safety and occupational health behaviors.

Alpha-synuclein cooperates with CSPalpha in preventing neurodegeneration.

Alpha-synuclein and cysteine-string protein-alpha (CSPalpha) are abundant synaptic vesicle proteins independently linked to neurodegeneration. Dominantly inherited mutations in alpha-synuclein cause Parkinson's disease, but the physiological role of alpha-synuclein remains unknown. Deletion of CSPalpha produces rapidly progressive neurodegeneration in mice, presumably because the cochaperone function of CSPalpha is essential for neuronal survival. Here, we report the surprising finding that transgenic expression of alpha-synuclein abolishes the lethality and neurodegeneration caused by deletion of CSPalpha. Conversely, ablation of endogenous synucleins exacerbates these phenotypes. Deletion of CSPalpha inhibits SNARE complex assembly; transgenic alpha-synuclein ameliorates this inhibition. In preventing neurodegeneration in CSPalpha-deficient mice, alpha-synuclein does not simply substitute for CSPalpha but acts by a downstream mechanism that requires phospholipid binding by alpha-synuclein. These observations reveal a powerful in vivo activity of alpha-synuclein in protecting nerve terminals against injury and suggest that this activity operates in conjunction with CSPalpha and SNARE proteins on the presynaptic membrane interface.