Targeting increased levels of APP in Down syndrome: Posiphen-mediated reductions in APP and its products reverse endosomal phenotypes in the Ts65Dn mouse model.

OBJECTIVE: Recent clinical trials targeting amyloid beta (Aß) and tau in Alzheimer's disease (AD) have yet to demonstrate efficacy. Reviewing the hypotheses for AD pathogenesis and defining possible links between them may enhance insights into both upstream initiating events and downstream mechanisms, thereby promoting discovery of novel treatments. Evidence that in Down syndrome (DS), a population markedly predisposed to develop early onset AD, increased APP gene dose is necessary for both AD neuropathology and dementia points to normalization of the levels of the amyloid precursor protein (APP) and its products as a route to further define AD pathogenesis and discovering novel treatments. BACKGROUND: AD and DS share several characteristic manifestations. DS is caused by trisomy of whole or part of chromosome 21; this chromosome contains about 233 protein-coding genes, including APP. Recent evidence points to a defining role for increased expression of the gene for APP and for its 99 amino acid C-terminal fragment (C99, also known as ß-CTF) in dysregulating the endosomal/lysosomal system. The latter is critical for normal cellular function and in neurons for transmitting neurotrophic signals. NEW/UPDATED HYPOTHESIS: We hypothesize that the increase in APP gene dose in DS initiates a process in which increased levels of full-length APP (fl-APP) and its products, including ß-CTF and possibly Aß peptides (Aß42 and Aß40), drive AD pathogenesis through an endosome-dependent mechanism(s), which compromises transport of neurotrophic signals. To test this hypothesis, we carried out studies in the Ts65Dn mouse model of DS and examined the effects of Posiphen, an orally available small molecule shown in prior studies to reduce fl-APP. In vitro, Posiphen lowered fl-APP and its C-terminal fragments, reversed Rab5 hyperactivation and early endosome enlargement, and restored retrograde transport of neurotrophin signaling. In vivo, Posiphen treatment (50 mg/kg/d, 26 days, intraperitoneal [i.p.]) of Ts65Dn mice was well tolerated and demonstrated no adverse effects in behavior. Treatment resulted in normalization of the levels of fl-APP, C-terminal fragments and small reductions in Aß species, restoration to normal levels of Rab5 activity, reduced phosphorylated tau (p-tau), and reversed deficits in TrkB (tropomyosin receptor kinase B) activation and in the Akt (protein kinase B [PKB]), ERK (extracellular signal-regulated kinase), and CREB (cAMP response element-binding protein) signaling pathways. Remarkably, Posiphen treatment also restored the level of choline acetyltransferase protein to 2N levels. These findings support the APP gene dose hypothesis, point to the need for additional studies to explore the mechanisms by which increased APP gene expression acts to increase the risk for AD in DS, and to possible utility of treatments to normalize the levels of APP and its products for preventing AD in those with DS. MAJOR CHALLENGES FOR THE HYPOTHESIS: Important unanswered questions are: (1) When should one intervene in those with DS; (2) would an APP-based strategy have untoward consequences on possible adaptive changes induced by chronically increased APP gene dose; (3) do other genes present on chromosome 21, or on other chromosomes whose expression is dysregulated in DS, contribute to AD pathogenesis; and (4) can one model strategies that combine the use of an APP-based treatment with those directed at other AD phenotypes including p-tau and inflammation. LINKAGE TO OTHER MAJOR THEORIES: The APP gene dose hypothesis interfaces with the amyloid cascade hypothesis of AD as well as with the genetic and cell biological observations that support it. Moreover, upregulation of fl-APP protein and products may drive downstream events that dysregulate tau homeostasis and inflammatory responses that contribute to propagation of AD pathogenesis.

Standards for preclinical research and publications in developmental anaesthetic neurotoxicity: expert opinion statement from the SmartTots preclinical working group.

In March 2019, SmartTots, a public-private partnership between the US Food and Drug Administration and the International Anesthesia Research Society, hosted a meeting attended by research experts, anaesthesia journal editors, and government agency representatives to discuss the continued need for rigorous preclinical research and the importance of establishing reporting standards for the field of anaesthetic perinatal neurotoxicity. This group affirmed the importance of preclinical research in the field, and welcomed novel and mechanistic approaches to answer some of the field's largest questions. The attendees concluded that summarising the benefits and disadvantages of specific model systems, and providing guidance for reporting results, would be helpful for designing new experiments and interpreting results across laboratories. This expert opinion report is a summary of these discussions, and includes a focused review of current animal models and reporting standards for the field of perinatal anaesthetic neurotoxicity. This will serve as a practical guide and road map for novel and rigorous experimental work.

Swedish Nerve Growth Factor Mutation (NGFR100W) Defines a Role for TrkA and p75NTR in Nociception.

Nerve growth factor (NGF) exerts multiple functions on target neurons throughout development. The recent discovery of a point mutation leading to a change from arginine to tryptophan at residue 100 in the mature NGFß sequence (NGFR100W) in patients with hereditary sensory and autonomic neuropathy type V (HSAN V) made it possible to distinguish the signaling mechanisms that lead to two functionally different outcomes of NGF: trophic versus nociceptive. We performed extensive biochemical, cellular, and live-imaging experiments to examine the binding and signaling properties of NGFR100W Our results show that, similar to the wild-type NGF (wtNGF), the naturally occurring NGFR100W mutant was capable of binding to and activating the TrkA receptor and its downstream signaling pathways to support neuronal survival and differentiation. However, NGFR100W failed to bind and stimulate the 75 kDa neurotrophic factor receptor (p75NTR)-mediated signaling cascades (i.e., the RhoA-Cofilin pathway). Intraplantar injection of NGFR100W into adult rats induced neither TrkA-mediated thermal nor mechanical acute hyperalgesia, but retained the ability to induce chronic hyperalgesia based on agonism for TrkA signaling. Together, our studies provide evidence that NGFR100W retains trophic support capability through TrkA and one aspect of its nociceptive signaling, but fails to engage p75NTR signaling pathways. Our findings suggest that wtNGF acts via TrkA to regulate the delayed priming of nociceptive responses. The integration of both TrkA and p75NTR signaling thus appears to regulate neuroplastic effects of NGF in peripheral nociception.SIGNIFICANCE STATEMENT In the present study, we characterized the naturally occurring nerve growth factor NGFR100W mutant that is associated with hereditary sensory and autonomic neuropathy type V. We have demonstrated for the first time that NGFR100W retains trophic support capability through TrkA, but fails to engage p75NTR signaling pathways. Furthermore, after intraplantar injection into adult rats, NGFR100W induced neither thermal nor mechanical acute hyperalgesia, but retained the ability to induce chronic hyperalgesia. We have also provided evidence that the integration of both TrkA- and p75NTR-mediated signaling appears to regulate neuroplastic effects of NGF in peripheral nociception. Our study with NGFR100W suggests that it is possible to uncouple trophic effect from nociceptive function, both induced by wild-type NGF.

Pathophysiology Associated with Traumatic Brain Injury: Current Treatments and Potential Novel Therapeutics.

Traumatic brain injury (TBI) is one of the leading causes of death of young people in the developed world. In the United States alone, 1.7 million traumatic events occur annually accounting for 50,000 deaths. The etiology of TBI includes traffic accidents, falls, gunshot wounds, sports, and combat-related events. TBI severity ranges from mild to severe. TBI can induce subtle changes in molecular signaling, alterations in cellular structure and function, and/or primary tissue injury, such as contusion, hemorrhage, and diffuse axonal injury. TBI results in blood-brain barrier (BBB) damage and leakage, which allows for increased extravasation of immune cells (i.e., increased neuroinflammation). BBB dysfunction and impaired homeostasis contribute to secondary injury that occurs from hours to days to months after the initial trauma. This delayed nature of the secondary injury suggests a potential therapeutic window. The focus of this article is on the (1) pathophysiology of TBI and (2) potential therapies that include biologics (stem cells, gene therapy, peptides), pharmacological (anti-inflammatory, antiepileptic, progrowth), and noninvasive (exercise, transcranial magnetic stimulation). In final, the review briefly discusses membrane/lipid rafts (MLR) and the MLR-associated protein caveolin (Cav). Interventions that increase Cav-1, MLR formation, and MLR recruitment of growth-promoting signaling components may augment the efficacy of pharmacologic agents or already existing endogenous neurotransmitters and neurotrophins that converge upon progrowth signaling cascades resulting in improved neuronal function after injury.

Amyloid precursor protein-mediated endocytic pathway disruption induces axonal dysfunction and neurodegeneration.

The endosome/lysosome pathway is disrupted early in the course of both Alzheimer's disease (AD) and Down syndrome (DS); however, it is not clear how dysfunction in this pathway influences the development of these diseases. Herein, we explored the cellular and molecular mechanisms by which endosomal dysfunction contributes to the pathogenesis of AD and DS. We determined that full-length amyloid precursor protein (APP) and its ß-C-terminal fragment (ß-CTF) act though increased activation of Rab5 to cause enlargement of early endosomes and to disrupt retrograde axonal trafficking of nerve growth factor (NGF) signals. The functional impacts of APP and its various products were investigated in PC12 cells, cultured rat basal forebrain cholinergic neurons (BFCNs), and BFCNs from a mouse model of DS. We found that the full-length wild-type APP (APPWT) and ß-CTF both induced endosomal enlargement and disrupted NGF signaling and axonal trafficking. ß-CTF alone induced atrophy of BFCNs that was rescued by the dominant-negative Rab5 mutant, Rab5S34N. Moreover, expression of a dominant-negative Rab5 construct markedly reduced APP-induced axonal blockage in Drosophila. Therefore, increased APP and/or ß-CTF impact the endocytic pathway to disrupt NGF trafficking and signaling, resulting in trophic deficits in BFCNs. Our data strongly support the emerging concept that dysregulation of Rab5 activity contributes importantly to early pathogenesis of AD and DS.

An Anti-ß-Amyloid Vaccine for Treating Cognitive Deficits in a Mouse Model of Down Syndrome.

In Down syndrome (DS) or trisomy of chromosome 21, the ß-amyloid (Aß) peptide product of the amyloid precursor protein (APP) is present in excess. Evidence points to increased APP gene dose and Aß as playing a critical role in cognitive difficulties experienced by people with DS. Particularly, Aß is linked to the late-life emergence of dementia as associated with neuropathological markers of Alzheimer's disease (AD). At present, no treatment targets Aß-related pathogenesis in people with DS. Herein we used a vaccine containing the Aß 1-15 peptide embedded into liposomes together with the adjuvant monophosphoryl lipid A (MPLA). Ts65Dn mice, a model of DS, were immunized with the anti-Aß vaccine at 5 months of age and were examined for cognitive measures at 8 months of age. The status of basal forebrain cholinergic neurons and brain levels of APP and its proteolytic products were measured. Immunization of Ts65Dn mice resulted in robust anti-Aß IgG titers, demonstrating the ability of the vaccine to break self-tolerance. The vaccine-induced antibodies reacted with Aß without detectable binding to either APP or its C-terminal fragments. Vaccination of Ts65Dn mice resulted in a modest, but non-significant reduction in brain Aß levels relative to vehicle-treated Ts65Dn mice, resulting in similar levels of Aß as diploid (2N) mice. Importantly, vaccinated Ts65Dn mice showed resolution of memory deficits in the novel object recognition and contextual fear conditioning tests, as well as reduction of cholinergic neuron atrophy. No treatment adverse effects were observed; vaccine did not result in inflammation, cellular infiltration, or hemorrhage. These data are the first to show that an anti-Aß immunotherapeutic approach may act to target Aß-related pathology in a mouse model of DS.

Membrane lipid rafts and neurobiology: age-related changes in membrane lipids and loss of neuronal function.

A better understanding of the cellular physiological role that plasma membrane lipids, fatty acids and sterols play in various cellular systems may yield more insight into how cellular and whole organ function is altered during the ageing process. Membrane lipid rafts (MLRs) within the plasma membrane of most cells serve as key organizers of intracellular signalling and tethering points of cytoskeletal components. MLRs are plasmalemmal microdomains enriched in sphingolipids, cholesterol and scaffolding proteins; they serve as a platform for signal transduction, cytoskeletal organization and vesicular trafficking. Within MLRs are the scaffolding and cholesterol binding proteins named caveolin (Cav). Cavs not only organize a multitude of receptors including neurotransmitter receptors (NMDA and AMPA receptors), signalling proteins that regulate the production of cAMP (G protein-coupled receptors, adenylyl cyclases, phosphodiesterases (PDEs)), and receptor tyrosine kinases involved in growth (Trk), but also interact with components that modulate actin and tubulin cytoskeletal dynamics (e.g. RhoGTPases and actin binding proteins). MLRs are essential for the regulation of the physiology of organs such as the brain, and age-related loss of cholesterol from the plasma membrane leads to loss of MLRs, decreased presynaptic vesicle fusion, and changes in neurotransmitter release, all of which contribute to different forms of neurodegeneration. Thus, MLRs provide an active membrane domain that tethers and reorganizes the cytoskeletal machinery necessary for membrane and cellular repair, and genetic interventions that restore MLRs to normal cellular levels may be exploited as potential therapeutic means to reverse the ageing and neurodegenerative processes.

The Effect of Clevidipine on Cerebral Blood Flow Velocity and Carbon Dioxide Reactivity in Human Volunteers.

BACKGROUND: Clevidipine is a short acting, esterase metabolized, calcium channel antagonist administered as a continuous infusion for control of hypertension. Its profile allows for rapid titration and may be uniquely suited to achieving tight hemodynamic targets in neurosurgical and neurocritical care patients. The present study was designed to investigate the effect of clevidipine infusion on cerebral blood flow and cerebral CO2 responsiveness as measured by cerebral blood flow velocity (CBFV) using transcranial Doppler. MATERIALS AND METHODS: CBFV was continuously recorded in 5 healthy subjects during the following conditions: baseline 1 (BL1); baseline with hyperventilation (HV1); baseline 2 (BL2); clevidipine infusion to achieve 15% mean arterial pressure (MAP) reduction (C15); clevidipine infusion to achieve 30% MAP reduction (C30); clevidipine infusion to 30% MAP reduction with hyperventilation (HV2). RESULTS: The mean CBFV during intermediate (C15) or maximum (C30) dose clevidipine infusion was unchanged compared with baseline (BL2) (F2,8=0.66; P=0.54). Cerebral CO2 reactivity, expressed as %[INCREMENT]CBFV/[INCREMENT]mm Hg CO2, was not significantly different in the presence of maximal-dose clevidipine (HV2) as compared with baseline (HV1) (1.6±0.4 vs. 1.6±0.3%[INCREMENT]CBFV/[INCREMENT]mm Hg CO2, P=0.73). CONCLUSIONS: Clevidipine infusion did not significantly increase CBFV nor was cerebral CO2 reactivity reduced during maximal-dose clevidipine infusion. Further systematic investigation of clevidipine in patients with central nervous system pathology seems justified.

A γ-secretase inhibitor, but not a γ-secretase modulator, induced defects in BDNF axonal trafficking and signaling: evidence for a role for APP.

Clues to Alzheimer disease (AD) pathogenesis come from a variety of different sources including studies of clinical and neuropathological features, biomarkers, genomics and animal and cellular models. An important role for amyloid precursor protein (APP) and its processing has emerged and considerable interest has been directed at the hypothesis that Aß peptides induce changes central to pathogenesis. Accordingly, molecules that reduce the levels of Aß peptides have been discovered such as γ-secretase inhibitors (GSIs) and modulators (GSMs). GSIs and GSMs reduce Aß levels through very different mechanisms. However, GSIs, but not GSMs, markedly increase the levels of APP CTFs that are increasingly viewed as disrupting neuronal function. Here, we evaluated the effects of GSIs and GSMs on a number of neuronal phenotypes possibly relevant to their use in treatment of AD. We report that GSI disrupted retrograde axonal trafficking of brain-derived neurotrophic factor (BDNF), suppressed BDNF-induced downstream signaling pathways and induced changes in the distribution within neuronal processes of mitochondria and synaptic vesicles. In contrast, treatment with a novel class of GSMs had no significant effect on these measures. Since knockdown of APP by specific siRNA prevented GSI-induced changes in BDNF axonal trafficking and signaling, we concluded that GSI effects on APP processing were responsible, at least in part, for BDNF trafficking and signaling deficits. Our findings argue that with respect to anti-amyloid treatments, even an APP-specific GSI may have deleterious effects and GSMs may serve as a better alternative.

Real-time imaging of axonal transport of quantum dot-labeled BDNF in primary neurons.

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are increasingly viewed as an early feature of neurodegenerative diseases, including Alzheimer's disease (AD) and Huntington's disease (HD). As yet unclear is the mechanism(s) by which axonal injury is induced. We reported the development of a novel technique to produce biologically active, monobiotinylated BDNF (mBtBDNF) that can be used to trace axonal transport of BDNF. Quantum dot-labeled BDNF (QD-BDNF) was produced by conjugating quantum dot 655 to mBtBDNF. A microfluidic device was used to isolate axons from neuron cell bodies. Addition of QD-BDNF to the axonal compartment allowed live imaging of BDNF transport in axons. We demonstrated that QD-BDNF moved essentially exclusively retrogradely, with very few pauses, at a moving velocity of around 1.06 µm/sec. This system can be used to investigate mechanisms of disrupted axonal function in AD or HD, as well as other degenerative disorders.

Aging and intellectual disability: insights from mouse models of Down syndrome.

Down syndrome (DS) is one of many causes of intellectual disability (ID), others including but not limited to, fetal alcohol syndrome, Fragile X syndrome, Rett syndrome, Williams syndrome, hypoxia, and infection. Down syndrome is characterized by a number of neurobiological problems resulting in learning and memory deficits and early onset Alzheimer's disease. The cognitive impairment in people with DS is virtually universal but varies considerably with respect to expressivity and severity. Significant advances in medical treatment and social inclusion have increased longevity in people with DS resulting in an increased aging population, thus highlighting the significance of early onset of dementia and the importance of identifying pharmacotherapies to treat DS-associated health complications in adults. Given its prevalence and established mouse models, this review will focus on ID in the DS population; specifically, the superimposed effect of aging on the complications already manifest in DS adults and the cognitive insights gained from studies on mouse models of DS.

Cognitive and pharmacological insights from the Ts65Dn mouse model of Down syndrome.

Down syndrome (DS) is a multi-faceted condition resulting in the most common genetic form of intellectual disability. Mouse models of DS, especially the Ts65Dn model, have been pivotal in furthering our understanding of the genetic, molecular and neurobiological mechanisms that underlie learning and memory impairments in DS. Cognitive and pharmacological insights from the Ts65Dn mouse model have led to remarkable translational progress in the development of therapeutic targets and in the emergence of DS clinical trials. Unravelling the pathogenic role of trisomic genes on human chromosome 21 and the genotype-phenotype relationship still remains a pertinent goal for tackling cognitive deficits in DS.

Propofol neurotoxicity is mediated by p75 neurotrophin receptor activation.

BACKGROUND: Propofol exposure to neurons during synaptogenesis results in apoptosis, leading to cognitive dysfunction in adulthood. Previous work from our laboratory showed that isoflurane neurotoxicity occurs through p75 neurotrophin receptor (p75(NTR)) and subsequent cytoskeleton depolymerization. Given that isoflurane and propofol both suppress neuronal activity, we hypothesized that propofol also induces apoptosis in developing neurons through p75(NTR). METHODS: Days in vitro 5-7 neurons were exposed to propofol (3 µM) for 6 h and apoptosis was assessed by cleaved caspase-3 (Cl-Csp3) immunoblot and immunofluorescence microscopy. Primary neurons from p75(NTR-/-) mice or wild-type neurons were treated with propofol, with or without pretreatment with TAT-Pep5 (10 µM, 15 min), a specific p75(NTR) inhibitor. P75(NTR-/-) neurons were transfected for 72 h with a lentiviral vector containing the synapsin-driven p75(NTR) gene (Syn-p75(NTR)) or control vector (Syn-green fluorescent protein) before propofol. To confirm our in vitro findings, wild-type mice and p75(NTR-/-) mice (PND5) were pretreated with either TAT-Pep5 or TAT-ctrl followed by propofol for 6 h. RESULTS: Neurons exposed to propofol showed a significant increase in Cl-Csp3, an effect attenuated by TAT-Pep5 and hydroxyfasudil. Apoptosis was significantly attenuated in p75(NTR-/-) neurons. In p75(NTR-/-) neurons transfected with Syn-p75(NTR), propofol significantly increased Cl-Csp3 in comparison with Syn-green fluorescent protein-transfected p75(NTR-/-) neurons. Wild-type mice exposed to propofol exhibited increased Cl-Csp3 in the hippocampus, an effect attenuated by TAT-Pep5. By contrast, propofol did not induce apoptosis in p75(NTR-/-) mice. CONCLUSION: These results demonstrate that propofol induces apoptosis in developing neurons in vivo and in vitro and implicate a role for p75(NTR) and the downstream effector RhoA kinase.

Isoflurane neurotoxicity is mediated by p75NTR-RhoA activation and actin depolymerization.

BACKGROUND: The mechanisms by which isoflurane injured the developing brain are not clear. Recent work has demonstrated that it is mediated in part by activation of p75 neurotrophin receptor. This receptor activates RhoA, a small guanosine triphosphatase that can depolymerize actin. It is therefore conceivable that inhibition of RhoA or prevention of cytoskeletal depolymerization might attenuate isoflurane neurotoxicity. This study was conducted to test these hypotheses using primary cultured neurons and hippocampal slice cultures from neonatal mouse pups. METHODS: Primary neuron cultures (days in vitro, 4-7) and hippocampal slice cultures from postnatal day 4-7 mice were exposed to 1.4% isoflurane (4 h). Neurons were pretreated with TAT-Pep5, an intracellular inhibitor of p75 neurotrophin receptor, the cytoskeletal stabilizer jasplakinolide, or their corresponding vehicles. Hippocampal slice cultures were pretreated with TAT-Pep5 before isoflurane exposure. RhoA activation was evaluated by immunoblot. Cytoskeletal depolymerization and apoptosis were evaluated with immunofluorescence microscopy using drebrin and cleaved caspase-3 staining, respectively. RESULTS: RhoA activation was increased after 30 and 120 min of isoflurane exposure in neurons; TAT-Pep5 (10 µm) decreased isoflurane-mediated RhoA activation at both time intervals. Isoflurane decreased drebrin immunofluorescence and enhanced cleaved caspase-3 in neurons, effects that were attenuated by pretreatment with either jasplakinolide (1 µm) or TAT-Pep5. TAT-Pep5 attenuated the isoflurane-mediated decrease in phalloidin immunofluorescence. TAT-Pep5 significantly attenuated isoflurane-mediated loss of drebrin immunofluorescence in hippocampal slices. CONCLUSIONS: Isoflurane results in RhoA activation, cytoskeletal depolymerization, and apoptosis. Inhibition of RhoA activation or prevention of downstream actin depolymerization significantly attenuated isoflurane-mediated neurotoxicity in developing neurons.

Loss of caveolin-1 accelerates neurodegeneration and aging.

BACKGROUND: The aged brain exhibits a loss in gray matter and a decrease in spines and synaptic densities that may represent a sequela for neurodegenerative diseases such as Alzheimer's. Membrane/lipid rafts (MLR), discrete regions of the plasmalemma enriched in cholesterol, glycosphingolipids, and sphingomyelin, are essential for the development and stabilization of synapses. Caveolin-1 (Cav-1), a cholesterol binding protein organizes synaptic signaling components within MLR. It is unknown whether loss of synapses is dependent on an age-related loss of Cav-1 expression and whether this has implications for neurodegenerative diseases such as Alzheimer's disease. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed brains from young (Yg, 3-6 months), middle age (Md, 12 months), aged (Ag, >18 months), and young Cav-1 KO mice and show that localization of PSD-95, NR2A, NR2B, TrkBR, AMPAR, and Cav-1 to MLR is decreased in aged hippocampi. Young Cav-1 KO mice showed signs of premature neuronal aging and degeneration. Hippocampi synaptosomes from Cav-1 KO mice showed reduced PSD-95, NR2A, NR2B, and Cav-1, an inability to be protected against cerebral ischemia-reperfusion injury compared to young WT mice, increased Aß, P-Tau, and astrogliosis, decreased cerebrovascular volume compared to young WT mice. As with aged hippocampi, Cav-1 KO brains showed significantly reduced synapses. Neuron-targeted re-expression of Cav-1 in Cav-1 KO neurons in vitro decreased Aß expression. CONCLUSIONS: Therefore, Cav-1 represents a novel control point for healthy neuronal aging and loss of Cav-1 represents a non-mutational model for Alzheimer's disease.