Wade C. Narrow

Chronic Neuron-Specific Tumor Necrosis Factor-Alpha Expression Enhances the Local Inflammatory Environment Ultimately Leading to Neuronal Death in 3xTg-AD Mice

Inflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta, appear integral in initiating and/or propagating Alzheimers disease (AD)-associated pathogenesis. We have previously observed a significant increase in the number of mRNA transcripts encoding the pro-inflammatory cytokine TNF-alpha, which correlated to regionally enhanced microglial activation in the brains of triple transgenic mice (3xTg-AD) before the onset of overt amyloid pathology. In this study, we reveal that neurons serve as significant sources of TNF-alpha in 3xTg-AD mice. To further define the role of neuronally derived TNF-alpha during early AD-like pathology, a recombinant adeno-associated virus vector expressing TNF-alpha was stereotactically delivered to 2-month-old 3xTg-AD mice and non-transgenic control mice to produce sustained focal cytokine expression. At 6 months of age, 3xTg-AD mice exhibited evidence of enhanced intracellular levels of amyloid-beta and hyperphosphorylated tau, as well as microglial activation. At 12 months of age, both TNF receptor II and Jun-related mRNA levels were significantly enhanced, and peripheral cell infiltration and neuronal death were observed in 3xTg-AD mice, but not in non-transgenic mice. These data indicate that a pathological interaction exists between TNF-alpha and the AD-related transgene products in the brains of 3xTg-AD mice. Results presented here suggest that chronic neuronal TNF-alpha expression promotes inflammation and, ultimately, neuronal cell death in this AD mouse model, advocating the development of TNF-alpha-specific agents to subvert AD.

Regeneration of the MPTP-Lesioned Dopaminergic System after Convection-Enhanced Delivery of AAV2-GDNF

Clinical studies to date have failed to establish therapeutic benefit of glial cell-derived neurotrophic factor (GDNF) in Parkinsons disease (PD). In contrast to previous nonclinical neuroprotective reports, this study shows clinically relevant and long-lasting regeneration of the dopaminergic system in rhesus macaques lesioned with 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine 3–6 months before GDNF gene delivery (AAV2-GDNF). The observed progressive amelioration of functional deficits, recovery of dopamine, and regrowth of fibers to the striatal neuropil demonstrate that high GDNF expression in the putamen promotes restoration of the dopaminergic system in a primate model of advanced PD. Extensive distribution of GDNF within the putamen and transport to the severely lesioned substantia nigra, after convection-enhanced delivery of AAV2-GDNF into the putamen, indicates anterograde transport via striatonigral connections and is anticipated to occur in PD patients. Overall, these data demonstrate nonclinical neurorestoration after putaminal infusion of AAV2-GDNF and suggest that clinical investigation in PD patients is warranted.

Early Oligodendrocyte/Myelin Pathology in Alzheimer’s Disease Mice Constitutes a Novel Therapeutic Target

The detection of myelin disruptions in Alzheimers disease (AD)-affected brain raises the possibility that oligodendrocytes undergo pathophysiological assault over the protracted course of this neurodegenerative disease. Oligodendrocyte compromise arising from direct toxic effects imparted by pathological amyloid-beta peptides and/or through signals derived from degenerating neurons could play an important role in the disease process. We previously demonstrated that 3xTg-AD mice, which harbor the human amyloid precursor protein Swedish mutant transgene, presenilin knock-in mutation, and tau P301L mutant transgene, exhibit significant alterations in overall myelination patterns and oligodendrocyte status at time points preceding the appearance of amyloid and tau pathology. Herein, we demonstrate that Abeta(1-42) leads to increased caspase-3 expression and apoptotic cell death of both nondifferentiated and differentiated mouse oligodendrocyte precursor (mOP) cells in vitro. Through use of a recombinant adeno-associated virus serotype-2 (rAAV2) vector expressing an Abeta(1-42)-specific intracellular antibody (intrabody), oligodendrocyte and myelin marker expression, as well as myelin integrity, were restored in the vector-infused brain regions of 3xTg-AD mice. Overall, this work provides further insights into the impact of Abeta(1-42)-mediated toxicity on the temporal and spatial progression of subtle myelin disruption during the early presymptomatic stages of AD and may help to validate new therapeutic options designed to avert these early impairments.

Ablation of TNF-RI/RII Expression in Alzheimer's Disease Mice Leads to an Unexpected Enhancement of Pathology: Implications for Chronic Pan-TNF-α Suppressive Therapeutic Strategies in the Brain

Alzheimers disease (AD) is a progressive neurodegenerative disorder characterized by severe memory loss and cognitive impairment. Neuroinflammation, including the extensive production of pro-inflammatory molecules and the activation of microglia, has been implicated in the disease process. Tumor necrosis factor (TNF)-α, a prototypic pro-inflammatory cytokine, is elevated in AD, is neurotoxic, and colocalizes with amyloid plaques in AD animal models and human brains. We previously demonstrated that the expression of TNF-α is increased in AD mice at ages preceding the development of hallmark amyloid and tau pathological features and that long-term expression of this cytokine in these mice leads to marked neuronal death. Such observations suggest that TNF-α signaling promotes AD pathogenesis and that therapeutics suppressing this cytokines activity may be beneficial. To dissect TNF-α receptor signaling requirements in AD, we generated triple-transgenic AD mice (3xTg-AD) lacking both TNF-α receptor 1 (TNF-RI) and 2 (TNF-RII), 3xTg-ADxTNF-RI/RII knock out, the cognate receptors of TNF-α. These mice exhibit enhanced amyloid and tau-related pathological features by the age of 15 months, in stark contrast to age-matched 3xTg-AD counterparts. Moreover, 3xTg-ADxTNF-RI/RII knock out-derived primary microglia reveal reduced amyloid-β phagocytic marker expression and phagocytosis activity, indicating that intact TNF-α receptor signaling is critical for microglial-mediated uptake of extracellular amyloid-β peptide pools. Overall, our results demonstrate that globally ablated TNF receptor signaling exacerbates pathogenesis and argues against long-term use of pan-anti-TNF-α inhibitors for the treatment of AD.

An improved method for generating consistent soluble amyloid-beta oligomer preparations for in vitro neurotoxicity studies

Soluble Abeta oligomers are recognized as playing a key role in Alzheimers disease (AD) pathophysiology. Despite their significance, many investigators encounter difficulty generating reliable preparations for in vitro and in vivo experiments. Solutions of Abeta are often unstable and soluble conformer profiles inconsistent. In this study we describe detailed methods for preparing Abeta oligomers that are stable for several weeks and are enriched for low and high molecular weight oligomeric forms, including the 56-kDa form, a conformer implicated in AD-related cognitive impairment. We characterize their structural and functional properties using Western blot, dot blot, atomic force microscopy, Thioflavine T fluorescence, and primary neuronal culture toxicity assays. These synthetic preparations should prove valuable to many studying Abeta-mediated mechanisms underlying AD.

Interferon-γ Differentially Affects Alzheimer’s Disease Pathologies and Induces Neurogenesis in Triple Transgenic-AD Mice

Inflammatory processes, including the episodic and/ or chronic elaboration of cytokines, have been identified as playing key roles in a number of neurological disorders. Whether these activities impart a disease-resolving and/or contributory outcome depends at least in part on the disease context, stage of pathogenesis, and cellular milieu in which these factors are released. Interferon-gamma (IFNgamma) is one such cytokine that produces pleiotropic effects in the brain. It is protective by ensuring maintenance of virus latency after infection, yet deleterious by recruiting and activating microglia that secrete potentially damaging factors at sites of brain injury. Using the triple-transgenic mouse model of Alzheimers disease (3xTg-AD), which develops amyloid and tau pathologies in a pattern reminiscent of human Alzheimers disease, we initiated chronic intrahippocampal expression of IFNgamma through delivery of a serotype-1 recombinant adeno-associated virus vector (rAAV1-IFNgamma). Ten months of IFNgamma expression led to an increase in microglial activation, steady-state levels of proinflammatory cytokine and chemokine transcripts, and severity of amyloid-related pathology. In contrast, these rAAV1-IFNgamma-treated 3xTg-AD mice also exhibited diminished phospho-tau pathology and evidence of increased neurogenesis. Overall, IFNgamma mediates what seem to be diametrically opposed functions in the setting of AD-related neurodegeneration. Gaining an understanding as to how these apparently divergent functions are interrelated and controlled could elucidate new therapeutic strategies designed to harness the neuroprotective activity of IFNgamma.

Aβ-directed Single-chain Antibody Delivery Via a Serotype-1 AAV Vector Improves Learning Behavior and Pathology in Alzheimer's Disease Mice

Alzheimers disease (AD) is a progressive dementing disorder characterized by age-related amyloid-beta (Aβ) deposition, neurofibrillary tangles, and synapse and neuronal loss. It is widely recognized that Aβ is a principal pathogenic mediator of AD. Our goal was to develop an immunotherapeutic approach, which would specifically lead to the clearance and/or neutralization of Aβ in the triple transgenic mouse model (3xTg-AD). These mice develop the amyloid and tangle pathologies and synaptic dysfunction reminiscent of human AD. Using a human single-chain variable fragment (scFv) antibody phage display library, a novel scFv antibody specific to Aβ was isolated, its activity characterized in vitro, and its open reading frame subsequently cloned into a recombinant adeno-associated virus (rAAV) vector. Three-month-old 3xTg-AD mice were intrahippocampally infused with serotype-1 rAAV vectors encoding Aβ-scFv or a control vector using convection-enhanced delivery (CED). Mice receiving rAAV1-Aβ-scFv harbored lower levels of insoluble Aβ and hyperphosphorylated tau, and exhibited improved cognitive function as measured by the Morris Water Maze (MWM) spatial memory task. These results underscore the potential of gene-based passive vaccination for AD, and provide further rationale for the development of Aβ-targeting strategies for this debilitating disease.Alzheimers disease (AD) is a progressive dementing disorder characterized by age-related amyloid-beta (Abeta) deposition, neurofibrillary tangles, and synapse and neuronal loss. It is widely recognized that Abeta is a principal pathogenic mediator of AD. Our goal was to develop an immunotherapeutic approach, which would specifically lead to the clearance and/or neutralization of Abeta in the triple transgenic mouse model (3xTg-AD). These mice develop the amyloid and tangle pathologies and synaptic dysfunction reminiscent of human AD. Using a human single-chain variable fragment (scFv) antibody phage display library, a novel scFv antibody specific to Abeta was isolated, its activity characterized in vitro, and its open reading frame subsequently cloned into a recombinant adeno-associated virus (rAAV) vector. Three-month-old 3xTg-AD mice were intrahippocampally infused with serotype-1 rAAV vectors encoding Abeta-scFv or a control vector using convection-enhanced delivery (CED). Mice receiving rAAV1-Abeta-scFv harbored lower levels of insoluble Abeta and hyperphosphorylated tau, and exhibited improved cognitive function as measured by the Morris Water Maze (MWM) spatial memory task. These results underscore the potential of gene-based passive vaccination for AD, and provide further rationale for the development of Abeta-targeting strategies for this debilitating disease.

Generating Differentially Targeted Amyloid-β Specific Intrabodies as a Passive Vaccination Strategy for Alzheimer's Disease

Amyloid-β (Aβ) has been identified as a key component in Alzheimers disease (AD). Significant in vitro and human pathological data suggest that intraneuronal accumulation of Aβ peptides plays an early role in the neurodegenerative cascade. We hypothesized that targeting an antibody-based therapeutic to specifically abrogate intracellular Aβ accumulation could prevent or slow disease onset. Aβ42-specific intracellular antibodies (intrabodies) with and without an intracellular trafficking signal were engineered from a previously characterized single-chain variable fragment (scFv) antibody. The intrabodies, one with an endoplasmic reticulum (ER) targeting signal and one devoid of a targeting sequence, were assessed in cells harboring a doxycycline (Dox)-regulated mutant human amyloid precursor protein Swedish mutant (hAPPswe) transcription unit for their abilities to prevent Aβ peptide egress. Adeno-associated virus (AAV) vectors expressing the engineered intrabodies were administered to young adult 3xTg-AD mice, a model that develops amyloid and Tau pathologies, prior to the initial appearance of intraneuronal Aβ. Chronic expression of the ER-targeted intrabody (IB) led to partial clearance of Aβ42 deposits and interestingly, in reduced staining for a pathologic phospho-Tau epitope (Thr231). This approach may provide insights into the functional relevance of intraneuronal Aβ accumulation in early AD and potentially lead to the development of new therapeutics.Amyloid-beta (A beta) has been identified as a key component in Alzheimers disease (AD). Significant in vitro and human pathological data suggest that intraneuronal accumulation of A beta peptides plays an early role in the neurodegenerative cascade. We hypothesized that targeting an antibody-based therapeutic to specifically abrogate intracellular A beta accumulation could prevent or slow disease onset. A beta 42-specific intracellular antibodies (intrabodies) with and without an intracellular trafficking signal were engineered from a previously characterized single-chain variable fragment (scFv) antibody. The intrabodies, one with an endoplasmic reticulum (ER) targeting signal and one devoid of a targeting sequence, were assessed in cells harboring a doxycycline (Dox)-regulated mutant human amyloid precursor protein Swedish mutant (hAPP(swe)) transcription unit for their abilities to prevent A beta peptide egress. Adeno-associated virus (AAV) vectors expressing the engineered intrabodies were administered to young adult 3xTg-AD mice, a model that develops amyloid and Tau pathologies, prior to the initial appearance of intraneuronal A beta. Chronic expression of the ER-targeted intrabody (IB) led to partial clearance of A beta 42 deposits and interestingly, in reduced staining for a pathologic phospho-Tau epitope (Thr231). This approach may provide insights into the functional relevance of intraneuronal A beta accumulation in early AD and potentially lead to the development of new therapeutics.

Chronic Neuron- and Age-Selective Down-Regulation of TNF Receptor Expression in Triple-Transgenic Alzheimer Disease Mice Leads to Significant Modulation of Amyloid- and Tau-Related Pathologies

Neuroinflammation, through production of proinflammatory molecules and activated glial cells, is implicated in Alzheimers disease (AD) pathogenesis. One such proinflammatory mediator is tumor necrosis factor α (TNF-α), a multifunctional cytokine produced in excess and associated with amyloid β-driven inflammation and cognitive decline. Long-term global inhibition of TNF receptor type I (TNF-RI) and TNF-RII signaling without cell or stage specificity in triple-transgenic AD mice exacerbates hallmark amyloid and neurofibrillary tangle pathology. These observations revealed that long-term pan anti-TNF-α inhibition accelerates disease, cautions against long-term use of anti-TNF-α therapeutics for AD, and urges more selective regulation of TNF signaling. We used adeno-associated virus vector-delivered siRNAs to selectively knock down neuronal TNF-R signaling. We demonstrate divergent roles for neuronal TNF-RI and TNF-RII where loss of opposing TNF-RII leads to TNF-RI-mediated exacerbation of amyloid β and Tau pathology in aged triple-transgenic AD mice. Dampening of TNF-RII or TNF-RI+RII leads to a stage-independent increase in Iba-1-positive microglial staining, implying that neuronal TNF-RII may act nonautonomously on the microglial cell population. These results reveal that TNF-R signaling is complex, and it is unlikely that all cells and both receptors will respond positively to broad anti-TNF-α treatments at various stages of disease. In aggregate, these data further support the development of cell-, stage-, and/or receptor-specific anti-TNF-α therapeutics for AD.

An Alzheimer's disease-relevant presenilin-1 mutation augments amyloid-beta-induced oligodendrocyte dysfunction.

White matter pathology has been documented in the brains of familial Alzheimers disease (FAD)‐afflicted individuals during presymptomatic and preclinical stages of AD. How these defects in myelination integrity arise and what roles they may play in AD pathophysiology have yet to be fully elucidated. We previously demonstrated that triple‐transgenic AD (3xTg‐AD) mice, which harbor the human amyloid precursor Swedish mutation, presenilin‐1 M146V (PS1M146V) knock‐in mutation, and tauP301L mutation, exhibit myelin abnormalities analogous to FAD patients and that Aβ1‐42 contributes to these white matter deficits. Herein, we demonstrate that the PS1M146V mutation predisposes mouse oligodendrocyte precursor (mOP) cells to Aβ1‐42‐induced alterations in cell differentiation in vitro. Furthermore, PS1M146V expression compromised mOP cell function and MBP protein distribution, a process that is further aggravated with exposure to Aβ1‐42. We found that the myelination defect and MBP subcellular mislocalization triggered by PS1M146V and Aβ1‐42 can be effectively prevented by treatment with the GSK‐3β inhibitor, TWS119, thereby implicating GSK‐3β kinase activity in this pathogenic cascade. Overall, this work provides further mechanistic insights into PS1M146V and Aβ1‐42‐driven oligodendrocyte dysfunction andmyelin damage during early presymptomatic stages of AD, and provides a new target in oligodendrocytes for developing therapies designed to avert AD‐related white matter pathology.