Neurofilaments form flexible bundles during neuritogenesis in culture and in mature axons in situ.

Neurofilaments (NFs) undergo cation-dependent phospho-mediated associations with each other and other cytoskeletal elements that support axonal outgrowth. Progressive NF-NF associations generate a resident, bundled population that undergoes exchange with transporting NFs. We examined the properties of bundled NFs. Bundles did not always display a fully linear profile but curved and twisted at various points along the neurite length. Bundles retracted faster than neurites and retracted bundles did not expand following extraction with Triton, indicating that they coiled passively rather than due to pressure from the cell. Bundles consisted of helically wound NFs, which may provide flexibility necessary for turning of growing axons during pathfinding. Interactions between NFs and other cytoskeletal elements may be disrupted en masse during neurite retraction or regionally during remodeling. It is suggested that bundles within long axons that cannot be fully retracted into the soma could provide maintain proximal support yet still allow more distal flexibility for remodeling and changing direction during pathfinding.

Beneficial and Deleterious Impact of a Nutritional Supplementation for Inhibition of Proliferation of Neuroblastoma in Culture.

Neuroblastoma, a cancer of the sympathetic nervous system, primarily affects infants and children ≤10 yr of age. High-risk neuroblastoma is associated with low survival rates and increased risks of treatment-related side-effects. Therefore, effective treatments that increase survival and reduce adverse side-effects are crucial. Cucurbitacin E (CucE), a nutritional supplement shown to have potential as an alternative to chemotherapy, was investigated for potential impact on neuroblastoma alone and in combination with the standard chemotherapeutic agent, paclitaxel, (PAC). CucE and PAC each inhibited proliferation of murine neuroblastoma cells in culture. Combined treatment with CucE and PAC also induced morphological differentiation. However, both differentiation and antiproliferative effects were reversible. Consequently, while nutritional supplementation represents a potential therapeutic approach toward treatment of cancer, certain nutritional/chemotherapeutic combinations may induce transient rather than permanent effects. Transient inhibition of proliferation by nutritional supplementation could inadvertently protect carcinogenic cells from toxicity otherwise induced by a chemotherapeutic agent. Combinatorial treatments involving nutritional supplements should therefore be utilized with caution.

Divergent and convergent roles for kinases and phosphatases in neurofilament dynamics.

C-terminal neurofilament phosphorylation mediates cation-dependent self-association leading to neurofilament incorporation into the stationary axonal cytoskeleton. Multiple kinases phosphorylate the C-terminal domains of the heavy neurofilament subunit (NF-H), including cyclin-dependent protein kinase 5 (CDK5), mitogen-activated protein kinases (MAPKs), casein kinase 1 and 2 (CK1 and CK2) and glycogen synthase kinase 3ß (GSK3ß). The respective contributions of these kinases have been confounded because they phosphorylate multiple substrates in addition to neurofilaments and display extensive interaction. Herein, differentiated NB2a/d1 cells were transfected with constructs expressing GFP-tagged NF-H, isolated NF-H sidearms and NF-H lacking the distal-most 187 amino acids. Cultures were treated with roscovitine, PD98059, Li(+), D4476, tetrabromobenzotriazole and calyculin, which are active against CDK5, MKK1 (also known as MAP2K1), GSK3ß, CK1, CK2 and protein phosphatase 1 (PP1), respectively. Sequential phosphorylation by CDK5 and GSK3ß mediated the neurofilament-neurofilament associations. The MAPK pathway (i.e. MKK1 to ERK1/2) was found to downregulate GSK3ß, and CK1 activated PP1, both of which promoted axonal transport and restricted neurofilament-neurofilament associations to axonal neurites. The MAPK pathway and CDK5, but not CK1 and GSK3ß, inhibited neurofilament proteolysis. These findings indicate that phosphorylation of neurofilaments by the proline-directed MAPK pathway and CDK5 counterbalance the impact of phosphorylation of neurofilaments by the non-proline-directed CK1 and GSK3ß.

Environmental enrichment and social interaction improve cognitive function and decrease reactive oxidative species in normal adult mice.

Environmental stimulation and increased social interactions stimulate cognitive performance, while decrease in these parameters can exacerbate cognitive decline as a function of illness, injury, or age. We examined the impact of environmental stimulation and social interactions on cognitive performance in healthy adult C57B1/6J mice. Mice were housed for 1 month individually or in groups of three (to prevent or allow social interaction) in either a standard environment (SE) or an enlarged cage containing nesting material and items classically utilized to stimulate exploration and activity ("enriched environment"; EE). Cognitive performance was tested by Y maze navigation and Novel Object Recognition (NOR; which compares the relative amount of time mice spent investigating a novel vs. a familiar object). Mice maintained for 1 month under isolated conditions in the SE statistically declined in performance versus baseline in the Y maze (p < 0.02; ANOVA). Performance under all other conditions did not change from baseline. Maintenance in groups in the SE statistically improved NOR (p < 0.01), whereas maintenance in isolation in the SE did not alter performance from baseline. Maintenance in the EE statistically improved performance in NOR for mice housed in groups and individually (p < 0.01). Maintenance under isolated conditions slightly increased reactive oxygen/nitrogen species (ROS/RNS) in brain. Environmental enrichment did not influence ROS/RNS. These findings indicate that environmental and social enrichment can positively influence cognitive performance in healthy adult mice, and support the notion that proactive approaches may delay age-related cognitive decline.

Differential roles of kinesin and dynein in translocation of neurofilaments into axonal neurites.

Neurofilament (NF) subunits translocate within axons as short NFs, non-filamentous punctate structures ('puncta') and diffuse material that might comprise individual subunits and/or oligomers. Transport of NFs into and along axons is mediated by the microtubule (MT) motor proteins kinesin and dynein. Despite being characterized as a retrograde motor, dynein nevertheless participates in anterograde NF transport through associating with long MTs or the actin cortex through its cargo domain; relatively shorter MTs associated with the motor domain are then propelled in an anterograde direction, along with any linked NFs. Here, we show that inhibition of dynein function, through dynamitin overexpression or intracellular delivery of anti-dynein antibody, selectively reduced delivery of GFP-tagged short NFs into the axonal hillock, with a corresponding increase in the delivery of puncta, suggesting that dynein selectively delivered short NFs into axonal neurites. Nocodazole-mediated depletion of short MTs had the same effect. By contrast, intracellular delivery of anti-kinesin antibody inhibited anterograde transport of short NFs and puncta to an equal extent. These findings suggest that anterograde axonal transport of linear NFs is more dependent upon association with translocating MTs (which are themselves translocated by dynein) than is transport of NF puncta or oligomers.

Positive argument for debate in J Neural Transmission: Alzheimer's disease: are we intervening too late? Yes, by years if not decades.

The ongoing debate as to whether we are or are not early enough in treatment for Alzheimer's disease presents distinct vantage points. Points expressed range from stressing the need for early preventive measures to highlighting the failure of "alternative" therapies, and concluding that we are unfortunately doing all that we can at present. Herein, we stress the worth of nutritional intervention, and review why such studies are often inherently compromised. We conclude that considerable education is needed to advance lifestyle modifications early enough to obtain their optimal effect, and instead of positioning "classical" interventions against "alternative" interventions, the combinations of both may impart maximal benefit. The introduction of novel detection methods at the earliest indications of cognitive impairment may provide a window of opportunity for initiation of preventative approaches.

Neurofilament cross-bridging competes with kinesin-dependent association of neurofilaments with microtubules.

The phosphorylation of neurofilaments (NFs) has long been considered to regulate their axonal transport rate and in doing so to provide stability to mature axons. Axons contain a centrally situated ;bundle' of closely opposed phospho-NFs that display a high degree of NF-NF associations and phospho-epitopes, surrounded by less phosphorylated ;individual' NFs that are often associated with kinesin and microtubules (MTs). Bundled NFs transport substantially slower than the surrounding individual NFs and might represent a resident population that stabilizes axons and undergoes replacement by individual NFs. To examine this possibility, fractions enriched in bundled NFs and individual NFs were generated from mice and NB2a/d1 cells by sedimentation of cytoskeletons over a sucrose cushion. More kinesin was recovered within individual versus bundled NF fractions. Individual but not bundled NFs aligned with purified MTs under cell-free conditions. The percentage of NFs that aligned with MTs was increased by the addition of kinesin, and inhibited by anti-kinesin antibodies. Bundles dissociated following incubation with EGTA or alkaline phosphatase, generating individual NFs that retained or were depleted of phospho-epitopes, respectively. These dissociated NFs aligned with MTs at a level identical to those originally isolated as individual NFs regardless of phosphorylation state. EGTA-mediated dissociation of bundles was prevented and reversed by excess Ca(2+), whereas individual NFs did not associate in the presence of excess Ca(2+). These findings confirm that bundling competes with NF-MT association, and provide a mechanism by which C-terminal NF phosphorylation might indirectly contribute to the observed slowing in axonal transport of phospho-NFs.

Improvement of cognitive performance by a nutraceutical formulation: Underlying mechanisms revealed by laboratory studies.

Cognitive decline, decrease in neuronal function and neuronal loss that accompany normal aging and dementia are the result of multiple mechanisms, many of which involve oxidative stress. Herein, we review these various mechanisms and identify pharmacological and non-pharmacological approaches, including modification of diet, that may reduce the risk and progression of cognitive decline. The optimal degree of neuronal protection is derived by combinations of, rather than individual, compounds. Compounds that provide antioxidant protection are particularly effective at delaying or improving cognitive performance in the early stages of Mild Cognitive Impairment and Alzheimer's disease. Laboratory studies confirm alleviation of oxidative damage in brain tissue. Lifestyle modifications show a degree of efficacy and may augment pharmacological approaches. Unfortunately, oxidative damage and resultant accumulation of biomarkers of neuronal damage can precede cognitive decline by years to decades. This underscores the importance of optimization of dietary enrichment, antioxidant supplementation and other lifestyle modifications during aging even for individuals who are cognitively intact.

Inhibitory neurons modulate spontaneous signaling in cultured cortical neurons: density-dependent regulation of excitatory neuronal signaling.

Cortical neuronal activity depends on a balance between excitatory and inhibitory influences. Culturing of neurons on multi-electrode arrays (MEAs) has provided insight into the development and maintenance of neuronal networks. Herein, we seeded MEAs with murine embryonic cortical/hippocampal neurons at different densities (<150 or >1000 cells mm(-2)) and monitored resultant spontaneous signaling. Sparsely seeded cultures displayed a large number of bipolar, rapid, high-amplitude individual signals with no apparent temporal regularity. By contrast, densely seeded cultures instead displayed clusters of signals at regular intervals. These patterns were observed even within thinner and thicker areas of the same culture. GABAergic neurons (25% of total neurons in our cultures) mediated the differential signal patterns observed above, since addition of the inhibitory antagonist bicuculline to dense cultures and hippocampal slice cultures induced the signal pattern characteristic of sparse cultures. Sparsely seeded cultures likely lacked sufficient inhibitory neurons to modulate excitatory activity. Differential seeding of MEAs can provide a unique model for analyses of perturbation in the interaction between excitatory and inhibitory function during aging and neuropathological conditions where dysregulation of GABAergic neurons is a significant component.

Tau interferes with axonal neurite stabilization and cytoskeletal composition independently of its ability to associate with microtubules.

Tau impacts overall axonal transport particularly when overexpressed by interfering with translocation of kinesin along microtubules (MTs) and/or as a cargo of kinesin by outcompeting other kinesin cargo. To discern between which of these mechanisms was more robust during axonal outgrowth, we overexpressed phosphomimetic (E18; which is incapable of MT binding), phospho-null (A18) or wild-type (WT) full-length human tau conjugated to EGFP, the latter two of which bind MTs. Expression of WT and A18 displayed increased acetylated MTs and resistance to colchicine, while expression of E18 did not, indicating that E18 did not contribute to MT stabilization. Expression of all tau constructs reduced overall levels of neurofilaments (NFs) within axonal neurites, and distribution of NFs along neurite lengths. Since NFs are another prominent cargo of kinesin during axonal neurite outgrowth, this finding is consistent with WT, A18 and E18 inhibiting NF transport to the same extent by competing as cargo of kinesin. These findings indicate that tau can impair axonal transport independently of association with MTs in growing axonal neurites.

Dietary deficiency increases presenilin expression, gamma-secretase activity, and Abeta levels: potentiation by ApoE genotype and alleviation by S-adenosyl methionine.

Apolipoprotein E4 (ApoE4) is a risk factor for Alzheimer's disease (AD). Whether this risk arises from a deficient function of E4 or the lack of protection provided by E2 or E3 is unclear. Previous studies demonstrate that deprivation of folate and vitamin E, coupled with dietary iron as a pro-oxidant, for 1 month displayed increased presenilin 1 (PS-1) expression, gamma-secretase, and Abeta generation in mice lacking ApoE (ApoE-/- mice). While ApoE-/- mice are a model for ApoE deficiency, they may not reflect the entire range of consequences of E4 expression. We therefore compared herein the impact of the above deficient diet on mice expressing human E2, E3, or E4. As folate deficiency is accompanied by a decrease in the major methyl donor, S-adenosyl methionine (SAM), additional mice received the deficient diet plus SAM. E2 was more protective than murine ApoE or E3 and E4. Surprisingly, PS-1 and gamma-secretase were over-expressed in E3 to the same extent as in E4 even under a complete diet, and were not alleviated by SAM supplementation. Abeta increased only in E4 mice maintained under the complete diet, and was alleviated by SAM supplementation. These findings suggest dietary compromise can potentiate latent risk factors for AD.

Dietary supplementation with apple juice decreases endogenous amyloid-beta levels in murine brain.

Folate deficiency has been associated with age-related neurodegeneration. We demonstrate herein that dietary deficiency in folate and vitamin E, coupled pro-oxidant stress induced by dietary iron, increased amyloid-beta (Abeta) levels in normal adult mice. This increase was potentiated by apolipoprotein E (ApoE) deficiency as shown by treatment of transgenic mice homozygously lacking murine ApoE. Dietary supplementation with apple juice concentrate in drinking water alleviated the increase in Abeta for both mouse genotypes. These findings provide further evidence linking nutritional and genetic risk factors for age-related neurodegeneration, and underscore that dietary supplementation may be useful to augment therapeutic approaches.

Deprivation of folate and B12 increases neurodegeneration beyond that accompanying deprivation of either vitamin alone.

Increased homocysteine has in some cases been linked with an increased incidence of Alzheimer's disease and motor neuron disease. Folate or B12 deficiency increases homocysteine, but controversy exists as to whether their levels also correlate with either disorder. Since their presence within various dietary constituents may confound interpretation, we tested the impact of deprivation of either or both in the closed environment of neuronal cell cultures. Deprivation of either increased cytosolic calcium, reactive oxygen species, intracellular homocysteine, and apoptosis, but deprivation of both fostered substantially larger increases, supporting the notion that both are required for optimal neuroprotection.

Efficacy of a vitamin/nutriceutical formulation for moderate-stage to later-stage Alzheimer's disease: a placebo-controlled pilot study.

Recent studies demonstrated efficacy of a vitamin/ nutriceutical formulation (folate, vitamin B12, alpha-tocopherol, S-adenosyl methionine, N-acetyl cysteine, and acetyl-L-carnitine) for mild to moderate Alzheimer's disease. Herein, we tested the efficacy of this formulation in a small cohort of 12 institutionalized patients diagnosed with moderate-stage to later-stage Alzheimer's disease. Participants were randomly separated into treatment of placebo groups. Participants receiving the formulation demonstrated a clinically significant delay in decline in the Dementia Rating Scale and clock-drawing test as compared to those receiving placebo. Institutional caregivers reported approximately 30% improvement in the Neuropyschiatric Inventory and maintenance of performance in the Alzheimer's Disease Cooperative Study-Activities of Daily Living for more than 9 months. This formulation holds promise for delaying the decline in cognition, mood, and daily function that accompanies the progression of Alzheimer's disease, and may be particularly useful as a supplement for pharmacological approaches during later stages of this disorder. A larger trial is warranted.

Erratum: Relative Efficacy of a Nutritional Intervention on Cognitive Performance Across the Adult Lifespan and During Early Cognitive Decline.

[This corrects the article DOI: 10.3233/ADR-190124.].

Relative Efficacy of a Nutritional Intervention on Cognitive Performance Across the Adult Lifespan and During Early Cognitive Decline.

A nutritional supplement has maintained or improved cognitive performance for healthy adults and individuals with mild cognitive impairment (MCI). Performance varied between 93 healthy adults aged 18-73 years versus 43 aged 75-85 years and among individuals with MCI. Healthy adult performance was stratified by age and for MCI as "intact" or "impaired" (Dementia Rating Scale guidelines). Some older individuals performed as well as younger individuals. All intact individuals with MCI maintained baseline performance; only impaired individuals receiving the supplement maintained baseline performance. Variation among elderly individuals can preclude observation of efficacy. Supplementation may maintain rather than improve performance for some individuals.

Collaborative Mentoring for Retaining Secondary Biology Teachers.

Veteran biology teachers are at risk of leaving the classroom due to burnout, feeling uninspired, and overall job dissatisfaction. One way to keep veteran teachers engaged is through continued mentoring. Yet current mentoring programs vary in scope, often focus too heavily on one-to-one talk, with mentors serving as therapists, and generally fail to include veteran teachers. Considering this is not how schools operate, we argue active mentoring for veteran teachers is best when embedded into regular school practice. Collaborative mentoring, as we have termed it, pairs experienced high school teachers with other veteran colleagues, including university professors, in professional development activities centering on improving classroom practices. We believe that collaborative mentoring holds potential to meet the needs of all stakeholders-high school students for support in learning laboratory and writing skills; university faculty for hands-on classroom work and reflective practice, as well as for sharing content and pedagogical knowledge with professionals in the field; and, specifically, veteran biology teachers for expanding access to meaningful professional development opportunities. Focusing on applicable classroom pedagogy serves as a cost-effective model for professional development for veteran teachers, possibly increasing job satisfaction and teacher retention in high schools across the nation.

Regulation of neurofilament dynamics by phosphorylation.

Neurofilament (NF) phosphorylation has long been considered to regulate axonal transport rate and in doing so to provide stability to mature axons. Studies utilizing mice in which the C-terminal region of NF subunits (which contains the vast majority of phosphorylation sites) has been deleted has prompted an ongoing challenge to this hypothesis. We evaluate the collective evidence to date for and against a role for NF C-terminal phosphorylation in regulation of axonal transport and in providing structural support for axons, including some novel studies from our laboratory. We present a few suggestions for further experimentation in this area, and expand upon previous models for axonal NF dynamics. Finally, we address how C-terminal phosphorylation is regionally and temporally regulated by a balance of kinase and phosphatase activities, and how misregulation of this balance can contribute to motor neuron disease.

S-adenosyl methionine: a natural therapeutic agent effective against multiple hallmarks and risk factors associated with Alzheimer's disease.

Recent preclinical and clinical findings demonstrate that dietary supplementation with S-adenosyl methionine alleviates a variety of risk factors and hallmarks associated with Alzheimer's disease. These findings support and extend prior studies, some of which are decades old, and support the notion that nutritional supplementation may represent an important augmentation for therapy in Alzheimer's disease.

S-adenosylmethionine mediates glutathione efficacy by increasing glutathione S-transferase activity: implications for S-adenosyl methionine as a neuroprotective dietary supplement.

When maintained on a folate-deficient, iron-rich diet, transgenic mice lacking in apolipoprotein E (ApoE-/- mice) demonstrate impaired activity of glutathione S-transferase (GST), resulting in increased oxidative species within brain tissue despite abnormally high levels of glutathione. These mice also exhibit reduced levels of S-adenosyl methionine (SAM) and increased levels of its hydrolysis product S-adenosyl homocysteine, which inhibits SAM usage. Supplementation of the above diet with SAM restored GST activity and eliminated reactive oxygen species at the expense of stockpiled glutathione, suggesting that one or more SAM-dependent reactions were required to maintain GST activity. We examined herein the impact of SAM on GST activity using a cell-free assay. SAM stimulated GST activity in a dose-response manner when added to homogenates derived from the above ApoE-/- mice. SAM also increased activity of purified rat liver GST and recombinant GST. Filtering of SAM through a 4 kDa cutoff and systematic withholding of reaction components eliminated the possibility of any additional contaminating enzyme. These findings confirm that SAM can exert a direct effect on GST activity. Since Alzheimer's disease is accompanied by reduced GST activity, diminished SAM and increased SAH, these findings underscore the critical role of SAM in maintenance of neuronal health.