Supplementation with apple juice can compensate for folate deficiency in a mouse model deficient in methylene tetrahydrofolate reductase activity.

Folate insufficiency promotes developmental as well as age-related disorders of the nervous system. The C677T variant of 5',10' methylene tetrahydrofolate reductase (MTHFR; which utilizes folate to regenerate methionine from homocysteine) displays reduced activity, and therefore promotes functional folate deficiency. Mice heterozygously lacking this gene (MTHFR+/- mice) represent a useful model for analysis of the impact of MTHFR deficiency and potential compensatory approaches. Since consumption of apple products has benefited mouse models subjected to dietary and/or genetically-induced folate deficiency, we compared the impact of supplementation with apple juice on cognitive and neuromuscular performance of mice MTHFR+/+ and +/- mice with and without dietary folate deficiency. Mice were maintained for 1 month on a standard, complete diet, or a challenge diet lacking folate, and vitamin E and containing a 50 g iron/500 g total diet as a pro-oxidant. Additional groups received apple juice concentrate (AJC) diluted to 0.5% (vol/vol) in their sole source of drinking water. MTHFR+/- mice demonstrated significantly impaired cognitive performance in standard reward-based T maze and the non-reward-based Y maze tests as compared to MTHFR+/+ when maintained on the complete diet; supplementation with AJC improved the performance of MTHFR+/- to the level observed for MTHFR+/+ mice. Maintenance for 1 month on the deficient diet reduced the performance of both genotypes in both tests, but supplementation with AJC prevented these reductions. MTHFR+/+ and +/- displayed virtually identical neuromuscular performance in the standard paw grip endurance test when maintained on the complete diet, and displayed similar, non-significant declines in performance when maintained on the deficient diet. Supplementation of either diet with AJC dramatically improved the performance of both genotypes. The findings presented herein indicate that supplementation with AJCs can compensate for genetic as well as dietary insufficiency in folate in a murine model of genetic folate compromise, and support the notion that dietary supplementation may be more critical under conditions of latent genetic compromise.

A vitamin/nutriceutical formulation improves memory and cognitive performance in community-dwelling adults without dementia.

Adults of both genders without dementia consumed a nutriceutical formulation ("NF," consisting of folic acid, B12, Vitamin E, S-adenosylmethionine, N-acetyl cysteine and Acetyl-L-carnitine), previously shown to improve cognitive performance in Alzheimer's disease, or placebo. Participants receiving NF but not placebo improved statistically and clinically in the California Verbal Learning Test II and the Trail-Making Test. Both groups improved further during a 3-month open-label extension. Additional individuals displayed identical improvement during a separate 6-month open-label trial. Performance declined to baseline following withdrawal of NF, and statistically improved when participants resumed taking NF. Additional participants receiving NF but not placebo demonstrated improvement within 2 weeks in Trail-making and Digit-Memory tests; both groups improved in a 2-week open-label extension. An increased percentage of participants > or = 74 years of age did not show improvement with NF, which may relate to age-related difficulties in adsorption and/or basal nutritional deficiencies, or age-related cognitive decline during the course of this study. These findings support the benefit of nutritional supplements for cognitive performance and suggest that additional supplementation may be required for the elderly.

Dietary and genetic compromise in folate availability reduces acetylcholine, cognitive performance and increases aggression: critical role of S-adenosyl methionine.

Folate deficiency has been associated with age-related neurodegeneration. One direct consequence of folate deficiency is a decline in the major methyl donor, S-adenosyl methionine (SAM). We demonstrate herein that pro-oxidant stress and dietary folate deficiency decreased levels of acetylcholine and impaired cognitive performance to various degrees in normal adult mice (9-12 months of age, adult mice heterozygously lacking 5',10'-methylene tetrahydrofolate reductase, homozygously lacking apolipoprotein E, or expressing human ApoE2, E3 or E4, and aged (2-2.5 year old) normal mice. Dietary supplementation with SAM in the absence of folate restored acetylcholine levels and cognitive performance to respective levels observed in the presence of folate. Increased aggressive behavior was observed among some but not all genotypes when maintained on the deficient diet, and was eliminated in all cases supplementation with SAM. Folate deficiency decreased levels of choline and N-methyl nicotinamide, while dietary supplementation with SAM increased methylation of nicotinamide to generate N-methyl nicotinamide and restored choline levels within brain tissue. Since N-methyl nicotinamide inhibits choline transport out of the central nervous system, and choline is utilized as an alternative methyl donor, these latter findings suggest that SAM may maintain acetylcholine levels in part by maintaining availability of choline. These findings suggest that dietary supplementation with SAM represents a useful therapeutic approach for age-related neurodegeneration which may augment pharmacological approaches to maintain acetylcholine levels, in particular during dietary or genetic compromise in folate usage.

S-adenosyl methionine: A connection between nutritional and genetic risk factors for neurodegeneration in Alzheimer's disease.

Clinical manifestation of Alzheimer's disease may depend upon interaction among its risk factors. Apolipoprotein E-deficient mice undergo oxidative damage and cognitive impairment when deprived of folate. We demonstrate herein that these mice were depleted in the methyl donor S-adenosyl methionine (SAM), which inhibited glutathione S-transferase, since this enzyme requires methylation of oxidative species prior to glutathione-dependent reduction. Dietary supplementation with SAM alleviated neuropathology. Since SAM deficiency promotes presenilin-1 overexpression, which increases gamma-secretase expression and Abeta generation, these findings directly link nutritional deficiency and genetic risk factors, and support supplementation with SAM for Alzheimer's therapy.

Dietary supplementation with apple juice concentrate alleviates the compensatory increase in glutathione synthase transcription and activity that accompanies dietary- and genetically-induced oxidative stress.

Increased oxidative stress, which can arise from dietary, environmental and/or genetic sources, contributes to the decline in cognitive performance during normal aging and in neurodegenerative conditions such as Alzheimer's disease. Supplementation with fruits and vegetables that are high in antioxidant potential can compensate for dietary and/or genetic deficiencies that promote increased oxidative stress. We have recently demonstrated that apple juice concentrate (AJC) prevents the increase in oxidative damage to brain tissue and decline in cognitive performance observed when transgenic mice lacking apolipoprotein E (ApoE-/-) are maintained on a vitamin-deficient diet and challenged with excess iron (included in the diet as a pro-oxidant). However, the mechanism by which AJC provided neuroprotection was not conclusively determined. Herein, we demonstrate that supplementation with AJC also prevents the compensatory increases in glutathione synthase transcription and activity that otherwise accompany maintenance of ApoE-/- mice on this vitamin-free diet in the presence of iron. Inclusion of the equivalent composition and concentration of sugars of AJC did not prevent these increases. These findings provide further evidence that the antioxidant potential of AJC can compensate for dietary and genetic deficiencies that otherwise promote neurodegeneration.

Apple juice prevents oxidative stress and impaired cognitive performance caused by genetic and dietary deficiencies in mice.

Increased oxidative stress contributes to the decline in cognitive performance during normal aging and in neurodegenerative conditions such as Alzheimer s disease. Dietary supplementation with fruits and vegetables that are high in antioxidant potential have in some cases compensated for dietary and/or genetic deficiencies that promote increased oxidative stress. Herein, we demonstrate that apple juice concentrate, administered ad libitum in drinking water, can compensate for the increased reactive oxygen species and decline in cognitive performance in maze trials observed when normal and transgenic mice lacking apolipoprotein E are deprived of folate and vitamin E. In addition, we demonstrate that this protective effect is not derived from the sugar content of the concentrate.

Efficacy of vitamin E, phosphatidyl choline and pyruvate on Abeta neurotoxicity in culture.

Oxidative stress is a pivotal factor in neuronal degeneration including that induced by exposure to amyloid-beta (Abeta). Treatment with antioxidants such as vitamin E can alleviate Abeta neurotoxicity. However, vitamin E was only marginally effective in clinical trials in Alzheimer's disease. Recent studies indicate that treatment with vitamin E (as a-tocopherol), sodium pyruvate and phosphatidyl choline (PC) is more effective than vitamin E alone against neuronal oxidative stress. We demonstrate herein that treatment of cultured murine cortical neurons with these 3 agents is also more effective than vitamin E alone against Abeta neurotoxicity as assayed by generation of reactive oxygen species and increased levels of phospho-isoforms of the microtubule-associated protein tau. These data underscore the potential efficacy of a combinatorial neuroprotective formulation against Abeta neurotoxicity.

Selective accumulation of the high molecular weight neurofilament subunit within the distal region of growing axonal neurites.

Axonal maturation in situ is accompanied by the transition of neurofilaments (NFs) comprised of only NF-M and NF-L to those also containing NF-H. Since NF-H participates in interactions of NFs with each other and with other cytoskeletal constituents, its appearance represents a critical event in the stabilization of axons that accompanies their maturation. Whether this transition is effected by replacement of "doublet" NFs with "triplet" NFs, or by incorporation of NF-H into existing doublet NFs is unclear. To address this issue, we examined the distribution of NF subunit immunoreactivity within axonal cytoskeletons of differentiated NB2a/d1 cell and DRG neurons between days 3-7 of outgrowth. Endogenous immunoreactivity either declined in a proximal-distal gradient or was relatively uniform along axons. This distribution was paralleled by microinjected biotinylated NF-L. By contrast, biotinylated NF-H displayed a bipolar distribution, with immunoreactivity concentrated within the proximal- and distal-most axonal regions. Proximal biotinylated NF-H accumulation paralleled that of endogenous NF immunoreactivity; however, distal-most biotinylated NF-H accumulation dramatically exceeded that of endogenous NFs and microinjected NF-L. This phenomenon was not due to co-polymerization of biotin-H with vimentin or alpha-internexin. This phenomenon declined with continued time in culture. These data suggest that NF-H can incorporate into existing cytoskeletal structures, and therefore suggest that this mechanism accounts for at least a portion of the accumulation of triplet NFs during axonal maturation. Selective NF-H accumulation into existing cytoskeletal structures within the distal-most region may provide de novo cytoskeletal stability for continued axon extension and/or stabilization.

Kinesin, dynein and neurofilament transport.

The recent demonstration that the fast axonal transport motors kinesin and dynein participate in axonal transport of neurofilaments--known to undergo slow transport--supports and extends recent studies indicating that some neurofilaments exhibit alternating bursts of fast axonal transport interspersed with periods of non-motility. In addition, these findings unify both certain aspects of axonal transport and neurofilament biology. We discuss these data herein in the context of both older and more recent studies of neurofilament dynamics.

Homocysteine potentiates beta-amyloid neurotoxicity: role of oxidative stress.

The cause of neuronal degeneration in Alzheimer's disease (AD) has not been completely clarified, but has been variously attributed to increases in cytosolic calcium and increased generation of reactive oxygen species (ROS). The beta-amyloid fragment (Abeta) of the amyloid precursor protein induces calcium influx, ROS and apoptosis. Homocysteine (HC), a neurotoxic amino acid that accumulates in neurological disorders including AD, also induces calcium influx and oxidative stress, which has been shown to enhance neuronal excitotoxicity, leading to apoptosis. We examined the possibility that HC may augment Abeta neurotoxicity. HC potentiated the Abeta-induced increase in cytosolic calcium and apoptosis in differentiated SH-SY-5Y human neuroblastoma cells. The antioxidant vitamin E and the glutathione precursor N-acetyl-L-cysteine blocked apoptosis following cotreatment with HC and Abeta, indicating that apoptosis is associated with oxidative stress. These findings underscore that moderate accumulation of excitotoxins at concentrations that alone do not appear to initiate adverse events may enhance the effects of other factors known to cause neurodegeneration such as Abeta.

Neurofilaments consist of distinct populations that can be distinguished by C-terminal phosphorylation, bundling, and axonal transport rate in growing axonal neurites.

We examined the steady-state distribution and axonal transport of neurofilament (NF) subunits within growing axonal neurites of NB2a/d1 cells. Ultrastructural analyses demonstrated a longitudinally oriented "bundle" of closely apposed NFs that was surrounded by more widely spaced individual NFs. NF bundles were recovered during fractionation and could be isolated from individual NFs by sedimentation through sucrose. Immunoreactivity toward the restrictive C-terminal phospho-dependent antibody RT97 was significantly more prominent on bundled than on individual NFs. Microinjected biotinylated NF subunits, GFP-tagged NF subunits expressed after transfection, and radiolabeled endogenous subunits all associated with individual NFs before they associated with bundled NFs. Biotinylated and GFP-tagged NF subunits did not accumulate uniformly along bundled NFs; they initially appeared within the proximal portion of the NF bundle and only subsequently were observed along the entire length of bundled NFs. These findings demonstrate that axonal NFs are not homogeneous but, rather, consist of distinct populations. One of these is characterized by less extensive C-terminal phosphorylation and a relative lack of NF-NF interactions. The other is characterized by more extensive C-terminal NF phosphorylation and increased NF-NF interactions and either undergoes markedly slower axonal transport or does not transport and undergoes turnover via subunit and/or filament exchange with individual NFs. Inhibition of phosphatase activities increased NF-NF interactions within living cells. These findings collectively suggest that C-terminal phosphorylation and NF-NF interactions are responsible for slowing NF axonal transport.

Differential efficacy of lipophilic and cytosolic antioxidants on generation of reactive oxygen species by amyloid-ß.

Exposure of neurons to amyloid-beta (Abeta) is accompanied by a cascade of oxidative damage that initiates with lipid peroxidation followed by subsequent generation of cytosolic free radicals and reactive oxygen species (ROS). The antioxidant vitamin E has been utilized to counteract Abeta-induced oxidative stress. We considered herein whether or not the lipid-solubility of vitamin E limits its neuroprotection to membrane-related oxidative damage, and renders it relatively ineffective where prior lipid peroxidation has already generated cytosolic free radicals and ROS. To test this possibility, we treated differentiated SH-SY-5Y human neuroblastoma with vitamin E or a cell-permeant antioxidant, N-acetyl cysteine (NAC), simultaneously with or 15 min after the application of Abeta. Both vitamin E and NAC prevented Abeta-induced ROS generation when applied simultaneously with Abeta, but only NAC prevented Abeta-induced ROS generation when added to cultures that had previously been exposed to Abeta. These results support the hypothesis that vitamin E can quench Abeta-induced lipid peroxidation, but cannot effectively quench ROS generated by prior lipid peroxidation. These findings in cell culture may provide limited insight into why vitamin E is not fully effective against neurodegeneration in AD in clinical settings, since some neuronal populations are likely to already have been compromised by prior Abeta exposure before vitamin E treatment was initiated.

The predominant form in which neurofilament subunits undergo axonal transport varies during axonal initiation, elongation, and maturation.

The forms in which neurofilament (NF) subunits undergo axonal transport is controversial. Recent studies from have provided real-time visualization of the slow axonal transport of NF subunits by transfecting neuronal cultures with constructs encoding green fluorescent protein (GFP)-conjugated NF-M subunits. In our studies in differentiated NB2a/d1 cells, the majority NF subunits underwent transport in the form of punctate NF precursors, while studies in cultured neurons have demonstrated transport of NF subunits in predominantly filamentous form. Although different constructs were used in these studies, transfection of the same cultured neurons with our construct yielded the filamentous pattern observed by others, while transfection of our cultures with their construct generated punctate structures, confirming that the observed differences did not reflect variances in assembly-competence among the constructs. Manipulation of intracellular kinase, phosphatase, and protease activities shifted the predominant form of GFP-conjugated subunits between punctate and filamentous, confirming, as shown previously for vimentin, that punctate structures represent precursors for intermediate filament formation. Since these prior studies were conducted at markedly differing neuronal differentiation states, we tested the alternate hypothesis that these differing results reflected developmental alterations in NF dynamics that accompany various stages of neuritogenesis. We conducted time-course analyses of transfected NB2a/d1 cells, including monitoring of transfected cells over several days, as well as transfecting cells at varying intervals prior to and following induction of differentiation and axonal neurite outgrowth. GFP-conjugated subunits were predominantly filamentous during the period of most robust axonal outgrowth and NF accumulation, and presented a mixed profile of punctate and filamentous forms prior to neuritogenesis and following the developmental slowing of neurite outgrowth. These analyses demonstrate that NF subunits are capable of undergoing axonal transport in multiple forms, and that the predominant form in which NF subunits undergo axonal transport varies in accord with the rate of axonal elongation and accumulation of NFs within developing axons.

Hypophosphorylated neurofilament subunits undergo axonal transport more rapidly than more extensively phosphorylated subunits in situ.

Axonal transport of neurofilaments (NFs) has long been considered to be regulated by phosphorylation. We present evidence that in optic axons of normal mice, the rate of NF axonal transport is inversely correlated with the NF phosphorylation state. In addition to 200 kDa NF-H and 145 kDa NF-M, axonal cytoskeletons from CNS contained a range of phospho-variants of NF-H migrating between 160-200 kDa, and of NF-M migrating at 97-145 kDa. While 160 kDa phospho-variants of NF-H have been well characterized, we confirmed the identity of the previously-described 97 kDa species as a hypophospho-variant of NF-M since (1) pulse-chase metabolic labeling confirmed the 97 kDa species to be a new synthesis product that was converted by phosphorylation over time into a form migrating at 145 kDa, (2) the 97 kDa protein reacted with multiple NF-M antibodies, including one specific for hypophosphorylated NF-M, and (3) dephosphorylation converted NF-M isoforms to 97 kDa. Autoradiographic analyses following metabolic radiolabeling demonstrated that hypophosphorylated NF-H and NF-M isoforms underwent substantially more rapid transport in situ than did extensively phosphorylated isoforms, while NF-H subunits bearing a developmentally delayed C-terminal phospho-epitope transported at a rate slower than that of total 200 kDa NF-H. Differential transport of phospho-variants also highlights that these variants are not homogeneously distributed among NFs, but are segregated to some extent among distinct, although probably overlapping, NF populations, indicating that axonal NFs are not homogeneous with respect to phosphorylation state.

Phosphorylation of tau alters its association with the plasma membrane.

1. The potential functions of the microtubule-associated protein tau have been expanded by the recent demonstration of its interaction with the plasma membrane. Since the association of tau with microtubules is regulated by phosphorylation, herein we examine whether or not the association of tau with the plasma membrane is also regulated by phosphorylation. 2. A range of tau isoforms migrating from 46 to 64 kDa was associated with crude particulate fractions derived from SH-SY-5Y human neuroblastoma cells, and were retained during the initial stages of plasma membrane purification. During the extensive washing utilized in purification of the plasma membrane, portions of each of these isoforms were depleted from the resultant purified membrane. Immunoblot analysis with phospho-dependent and -independent antibodies revealed selective depletion of phospho isoforms during membrane washing. This effect was more pronounced for the slowest-migrating (64-kDa) tau isoform. 3. This putative influence of phosphorylation on the association of tau with the plasma membrane was further probed by transfection of SH-SY-5Y human neuroblastoma cells with a tau construct that could associate with the plasma membrane but not with microtubules. Treatment with phorbol ester or calcium ionophore, both of which increased phospho-tau levels within the cytosol and plasma membrane, was accompanied by the dissociation of this tau construct from the membrane. 4. These data indicate that phosphorylation regulates the association with the plasma membrane. Dissociation from the membrane by phosphorylation may place tau at risk for hyperphosphorylation and ultimate PHF formation in a manner previously considered for tau dissociated from microtubules.

Phospho-dependent association of neurofilament proteins with kinesin in situ.

Recent studies demonstrate co-localization of kinesin with neurofilament (NF) subunits in culture and suggest that kinesin participates in NF subunit distribution. We sought to determine whether kinesin was also associated with NF subunits in situ. Axonal transport of NF subunits in mouse optic nerve was perturbed by the microtubule (MT)-depolymerizing drug vinblastine, indicating that NF transport was dependent upon MT dynamics. Kinesin co-precipitated during immunoprecipitation of NF subunits from optic nerve. The association of NFs and kinesin was regulated by NF phosphorylation, since (1) NF subunits bearing developmentally delayed phospho-epitopes did not co-purify in a microtubule motor preparation from CNS while less phosphorylated forms did; (2) subunits bearing these phospho-epitopes were selectively not co-precipitated with kinesin; and (3) phosphorylation under cell-free conditions diminished the association of NF subunits with kinesin. The nature and extent of this association was further examined by intravitreal injection of (35)S-methionine and monitoring NF subunit transport along optic axons. As previously described by several laboratories, the wave of NF subunits underwent a progressive broadening during continued transport. The front, but not the trail, of this broadening wave of NF subunits was co-precipitated with kinesin, indicating that (1) the fastest-moving NFs were associated with kinesin, and (2) that dissociation from kinesin may foster trailing of NF subunits during continued transport. These data suggest that kinesin participates in NF axonal transport either by directly translocating NFs and/or by linking NFs to transporting MTs. Both Triton-soluble as well as cytoskeleton-associated NF subunits were co-precipitated with kinesin; these data are considered in terms of the form(s) in which NF subunits undergo axonal transport.

Beta-amyloid-induced calcium influx induces apoptosis in culture by oxidative stress rather than tau phosphorylation.

Beta-amyloid (betaA) toxicity in culture is accompanied by multiple events culminating in apoptosis. Calcium influx may represent the initial event, since calcium chelation prevents all subsequent events, while subsequent events include increased generation of reactive oxygen species (ROS) and hyperphosphorylation of tau. In the present study, we undertook to determine whether ROS generation or tau hyperphosphorylation mediate betaA-induced apoptosis. The anti-oxidant vitamin E or the kinase inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenslfonamide (W7) was added following brief treatment of differentiated SH-SY-5Y human neuroblastoma cells with 22 microM betaA. Under these conditions, vitamin E prevented ROS generation and apoptosis, but did not prevent intracellular calcium accumulation or tau phosphorylation. W7 prevented tau phosphorylation but did not block betaA-induced calcium influx, ROS generation or apoptosis. While these studies do not address the long-term consequences of PHF formation, they indicate that ROS generation, rather than tau hyperphosphorylation, leads to apoptosis following betaA-induced calcium influx into cultured cells.

C-terminal phosphorylation of the high molecular weight neurofilament subunit correlates with decreased neurofilament axonal transport velocity.

We probed the relationship of NF axonal transport of neurofilaments (NFs) to their phosphorylation state by comparing these parameters in two closely-aged groups of young adult mice - 2 and 5 months of age. This particular time interval was selected since prior studies demonstrate that optic axons have already completed axonal caliber expansion and attained adult NF levels by 2 months but, as shown herein, continue to increase NF-H C-terminal phosphorylation. NF axonal transport was monitored by autoradiographic analysis of the distribution of radiolabeled subunits immunoprecipitated from optic axon segments at intervals following intravitreal injection of 35S-methionine. Both the peak and front of radiolabeled NFs translocated faster in 2- vs. 5-month-old mice. This developmental decline in NF transport rate was not due to reduced incorporation of NFs into the cytoskeleton, nor to an overall decline in slow axonal transport. By excluding or minimizing other factors, these findings support previous conclusions that C-terminal NF phosphorylation regulates NF axonal transport.

Axonal transport of microtubule-associated protein 1B (MAP1B) in the sciatic nerve of adult rat: distinct transport rates of different isoforms.

Cytoskeletal proteins are axonally transported with slow components a and b (SCa and SCb). In peripheral nerves, the transport velocity of SCa, which includes neurofilaments and tubulin, is 1-2 mm/d, whereas SCb, which includes actin, tubulin, and numerous soluble proteins, moves as a heterogeneous wave at 2-4 mm/d. We have shown that two isoforms of microtubule-associated protein 1B (MAP1B), which can be separated on SDS polyacrylamide gels on the basis of differences in their phosphorylation states (band I and band II), were transported at two different rates. All of band I MAP1B moved as a coherent wave at a velocity of 7-9 mm/d, distinct from slow axonal transport components SCa and SCb. Several other proteins were detected within the component that moved at the velocity of 7-9 mm/d, including the leading wave of tubulin and actin. The properties of this component define a distinct fraction of the slow axonal transport that we suggest to term slow component c (SCc). The relatively fast transport of the phosphorylated MAP1B isoform at 7-9 mm/d may account for the high concentration of phosphorylated MAP1B in the distal end of growing axons. In contrast to band I MAP1B, the transport profile of band II was complex and contained components moving with SCa and SCb and a leading edge at SCc. Thus, MAP1B isoforms in different phosphorylation states move with distinct components of slow axonal transport, possibly because of differences in their abilities to associate with other proteins.