Michael T. Maloney

β-Secretase-Cleaved Amyloid Precursor Protein Accumulates at Actin Inclusions Induced in Neurons by Stress or Amyloid β: A Feedforward Mechanism for Alzheimer's Disease

Rod-like inclusions (rods), composed of actin saturated with actin depolymerizing factor (ADF)/cofilin, are induced in hippocampal neurons by ATP depletion, oxidative stress, and excess glutamate and occur in close proximity to senile plaques in human Alzheimers disease (AD) brain (Minamide et al., 2000). Here, we show rods are found in brains from transgenic AD mice. Soluble forms of amyloid β (Aβ1–42) induce the formation of rods in a maximum of 19% of cultured hippocampal neurons in a time- and concentration-dependent manner. Approximately one-half of the responding neurons develop rods within 6 h or with as little as 10 nm Aβ1–42. Aβ1–42 induces the activation (dephosphorylation) of ADF/cofilin in neurons that form rods. Vesicles containing amyloid precursor protein (APP), β-amyloid cleavage enzyme, and presenilin-1, a component of the γ-secretase complex, accumulate at rods. The β-secretase-cleaved APP (either β-C-terminal fragment of APP or Aβ) also accumulates at rods. These results suggest that rods, formed in response to either Aβ or some other stress, block the transport of APP and enzymes involved in its processing to Aβ. These stalled vesicles may provide a site for producing Aβ1–42, which may in turn induce more rods in surrounding neurons, and expand the degenerative zone resulting in plaque formation.

Cofilin-mediated neurodegeneration in alzheimer’s disease and other amyloidopathies

Transport defects may arise in various neurodegenerative diseases from failures in molecular motors, microtubule abnormalities, and the chaperone/proteasomal degradation pathway leading to aggresomal-lysosomal accumulations. These defects represent important steps in the neurodegenerative cascade, although in many cases, a clear consensus has yet to be reached regarding their causal relationship to the disease. A growing body of evidence lends support to a link between neurite transport defects in the very early stages of many neurodegenerative diseases and alterations in the organization and dynamics of the actin cytoskeleton initiated by filament dynamizing proteins in the ADF/cofilin family. This article focuses on cofilin, which in neurons under stress, including stress induced by the amyloid-β (Aβ) 1–42 peptide, undergoes dephosphorylation (activation) and forms rod-shaped actin boundles (rods). Rods inhibit transport, are sites of amyloid precursor protein accumulation, and contribute to the pathology of Alzheimer’s disease. Because rods form rapidly in response to anoxia, they could also contribute to synaptic deficits associated with ischemic brain injury (e.g., stroke). Surprisingly, cofilin undergoes phosphorylation (inactivation) in hippocampal neurons treated with Aβ1–40 at high concentrations, and these neurons undergo dystrophic morphological changes, including accumulation of pretangle phosphorylated-τ. Therefore, extremes in phosphoregulation of cofilin by different forms of Aβ may explain much of the Alzheimer’s disease pathology and provide mechanisms for synaptic loss and plaque expansion.

Increased efficiency of the GABAA and GABAB receptor-mediated neurotransmission in the Ts65Dn mouse model of Down syndrome.

Cognitive impairment in Down syndrome (DS) involves the hippocampus. In the Ts65Dn mouse model of DS, deficits in hippocampus-dependent learning and synaptic plasticity were linked to enhanced inhibition. However, the mechanistic basis of changes in inhibitory efficiency remains largely unexplored, and efficiency of the GABAergic synaptic neurotransmission has not yet been investigated in direct electrophysiological experiments. To investigate this important feature of neurobiology of DS, we examined synaptic and molecular properties of the GABAergic system in the dentate gyrus (DG) of adult Ts65Dn mice. Both GABAA and GABAB receptor-mediated components of evoked inhibitory postsynaptic currents (IPSCs) were significantly increased in Ts65Dn vs. control (2N) DG granule cells. These changes were unaccompanied by alterations in hippocampal levels of GABAA (α1, α2, α3, α5 and γ2) or GABAB (Gbr1a and Gbr1b) receptor subunits. Immunoreactivity for GAD65, a marker for GABAergic terminals, was also unchanged. In contrast, there was a marked change in functional parameters of GABAergic synapses. Paired stimulations showed reduced paired-pulse ratios of both GABAA and GABAB receptor-mediated IPSC components (IPSC2/IPSC1), suggesting an increase in presynaptic release of GABA. Consistent with increased gene dose, the level of the Kir3.2 subunit of potassium channels, effectors for postsynaptic GABAB receptors, was increased. This change was associated with enhanced postsynaptic GABAB/Kir3.2 signaling following application of the GABAB receptor agonist baclofen. Thus, both GABAA and GABAB receptor-mediated synaptic efficiency is increased in the Ts65Dn DG, thus likely contributing to deficient synaptic plasticity and poor learning in DS.

Amyloid beta dimers/trimers potently induce cofilin-actin rods that are inhibited by maintaining cofilin-phosphorylation.

BackgroundPreviously we reported 1 μM synthetic human amyloid beta1-42 oligomers induced cofilin dephosphorylation (activation) and formation of cofilin-actin rods within rat hippocampal neurons primarily localized to the dentate gyrus.ResultsHere we demonstrate that a gel filtration fraction of 7PA2 cell-secreted SDS-stable human Aβ dimers and trimers (Aβd/t) induces maximal neuronal rod response at ~250 pM. This is 4,000-fold more active than traditionally prepared human Aβ oligomers, which contain SDS-stable trimers and tetramers, but are devoid of dimers. When incubated under tyrosine oxidizing conditions, synthetic human but not rodent Aβ1-42, the latter lacking tyrosine, acquires a marked increase (620 fold for EC50) in rod-inducing activity. Gel filtration of this preparation yielded two fractions containing SDS-stable dimers, trimers and tetramers. One, eluting at a similar volume to 7PA2 Aβd/t, had maximum activity at ~5 nM, whereas the other, eluting at the void volume (high-n state), lacked rod inducing activity at the same concentration. Fractions from 7PA2 medium containing Aβ monomers are not active, suggesting oxidized SDS-stable Aβ1-42 dimers in a low-n state are the most active rod-inducing species. Aβd/t-induced rods are predominantly localized to the dentate gyrus and mossy fiber tract, reach significance over controls within 2 h of treatment, and are reversible, disappearing by 24 h after Aβd/t washout. Overexpression of cofilin phosphatases increase rod formation when expressed alone and exacerbate rod formation when coupled with Aβd/t, whereas overexpression of a cofilin kinase inhibits Aβd/t-induced rod formation.ConclusionsTogether these data support a mechanism by which Aβd/t alters the actin cytoskeleton via effects on cofilin in neurons critical to learning and memory.

Mapping Cofilin-Actin Rods in Stressed Hippocampal Slices and the Role of cdc42 in Amyloid-beta-Induced Rods

Dissociated hippocampal neurons exposed to a variety of degenerative stimuli form neuritic cofilin-actin rods. Here we report on stimulus driven regional rod formation in organotypic hippocampal slices. Ultrastructural analysis of rods formed in slices demonstrates mitochondria and vesicles become entrapped within some rods. We developed a template for combining and mapping data from multiple slices, enabling statistical analysis for the identification of vulnerable sub-regions. Amyloid-beta (Abeta) induces rods predominantly in the dentate gyrus region, and Abeta-induced rods are reversible following washout. Rods that persist 24 h following transient (30 min) ATP-depletion are broadly distributed, whereas rods formed in response to excitotoxic glutamate localize within and nearby the pyramidal neurons. Time-lapse imaging of cofilin-GFP-expressing neurons within slices shows neuronal rod formation begins rapidly and peaks by 10 min of anoxia. In approximately 50% of responding neurons, Abeta-induced rod formation acts via cdc42, an upstream regulator of cofilin. These new observations support a role for cofilin-actin rods in stress-induced disruption of cargo transport and synaptic function within hippocampal neurons and suggest both cdc42-dependent and independent pathways modulate cofilin activity downstream from Abeta.

A novel method for producing mono-biotinylated, biologically active neurotrophic factors: An essential reagent for single molecule study of axonal transport

In this report, we describe a novel method for producing mature and biologically active mono-biotinylated nerve growth factors (mBtNGF) that can be used for single molecule studies of real-time movement of neurotrophins within axons of neurons. We inserted an AviTag sequence into the C-terminal of the full length mouse preproNGF cDNA and cloned the fusion construct into a pcDNA3.1 mammalian expression vector. We also subcloned the Escherichia coli biotin ligase, BirA, into a pcDNA3.1 vector. These two plasmids were then transiently co-expressed in HEK293FT cells. As a result, the AviTag located in the C-terminal of preproNGF was selectively ligated to a single biotin by BirA. The prepro sequence of NGF was subsequently cleaved within the cell. Mature mono-biotinylated NGF (mBtNGF) was secreted into cell culture media and was purified using Ni resin. We carried out activity assays and our results showed that mBtNGF retained biological activities that were comparable to normal NGF purified from mouse sub maxillary glands. We further verified the biotinylation efficiency of mBtNGF and the level of non-biotinylated NGF was virtually undetectable in the final preparation. Finally, by conjugating to quantum-dot streptavidin, mBtNGF was successfully used for single molecule study of axonal NGF trafficking in neurons.

Production and use of replication-deficient adenovirus for transgene expression in neurons.

Adenoviruses infect a wide range of cell types, do not require integration into the host cell genome, and can be produced as replication-deficient viruses capable of expressing transgenes behind any desired promoter. Thus, they are ideal for use in expressing transgenes in the postmitotic neuron. This chapter describes simplifications in the protocols for making recombinant adenoviruses and their use in expressing transgenes in primary neurons of several different types.

Calcium tips the balance: a microtubule plus end to lattice binding switch operates in the carboxyl terminus of BPAG1n4.

Microtubules (MTs) are integral to numerous cellular functions, such as cell adhesion, differentiation and intracellular transport. Their dynamics are largely controlled by diverse MT‐interacting proteins, but the signalling mechanisms that regulate these interactions remain elusive. In this report, we identify a rapid, calcium‐regulated switch between MT plus end interaction and lattice binding within the carboxyl terminus of BPAG1n4. This switch is EF‐hand dependent, and mutations of the EF‐hands abolish this dynamic behaviour. Our study thus uncovers a new, calcium‐dependent regulatory mechanism for a spectraplakin, BPAG1n4, at the MT plus end.

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

In Down syndrome (DS) or trisomy of chromosome 21, the β-amyloid (Aβ) peptide product of the amyloid precursor protein (APP) is present in excess. Evidence points to increased APP gene dose and Aβ as playing a critical role in cognitive difficulties experienced by people with DS. Particularly, Aβ is linked to the late-life emergence of dementia as associated with neuropathological markers of Alzheimer’s disease (AD). At present, no treatment targets Aβ–related pathogenesis in people with DS. Herein we used a vaccine containing the Aβ 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-Aβ 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-Aβ IgG titers, demonstrating the ability of the vaccine to break self-tolerance. The vaccine-induced antibodies reacted with Aβ 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 Aβ levels relative to vehicle-treated Ts65Dn mice, resulting in similar levels of Aβ 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-Aβ immunotherapeutic approach may act to target Aβ-related pathology in a mouse model of DS.

Nemitin, a Novel Map8/Map1s Interacting Protein with Wd40 Repeats

In neurons, a highly regulated microtubule cytoskeleton is essential for many cellular functions. These include axonal transport, regional specialization and synaptic function. Given the critical roles of microtubule-associated proteins (MAPs) in maintaining and regulating microtubule stability and dynamics, we sought to understand how this regulation is achieved. Here, we identify a novel LisH/WD40 repeat protein, tentatively named nemitin (neuronal enriched MAP interacting protein), as a potential regulator of MAP8-associated microtubule function. Based on expression at both the mRNA and protein levels, nemitin is enriched in the nervous system. Its protein expression is detected as early as embryonic day 11 and continues through adulthood. Interestingly, when expressed in non-neuronal cells, nemitin displays a diffuse pattern with puncta, although at the ultrastructural level it localizes along the microtubule network in vivo in sciatic nerves. These results suggest that the association of nemitin to microtubules may require an intermediary protein. Indeed, co-expression of nemitin with microtubule-associated protein 8 (MAP8) results in nemitin losing its diffuse pattern, instead decorating microtubules uniformly along with MAP8. Together, these results imply that nemitin may play an important role in regulating the neuronal cytoskeleton through an interaction with MAP8.