Rab12 is a regulator of LRRK2 and its activation by damaged lysosomes.

Leucine-rich repeat kinase 2 (LRRK2) variants associated with Parkinson's disease (PD) and Crohn's disease lead to increased phosphorylation of its Rab substrates. While it has been recently shown that perturbations in cellular homeostasis including lysosomal damage can increase LRRK2 activity and localization to lysosomes, the molecular mechanisms by which LRRK2 activity is regulated have remained poorly defined. We performed a targeted siRNA screen to identify regulators of LRRK2 activity and identified Rab12 as a novel modulator of LRRK2-dependent phosphorylation of one of its substrates, Rab10. Using a combination of imaging and immunopurification methods to isolate lysosomes, we demonstrated that Rab12 is actively recruited to damaged lysosomes and leads to a local and LRRK2-dependent increase in Rab10 phosphorylation. PD-linked variants, including LRRK2 R1441G and VPS35 D620N, lead to increased recruitment of LRRK2 to the lysosome and a local elevation in lysosomal levels of pT73 Rab10. Together, these data suggest a conserved mechanism by which Rab12, in response to damage or expression of PD-associated variants, facilitates the recruitment of LRRK2 and phosphorylation of its Rab substrate(s) at the lysosome.

Lysosomes are cellular compartments tasked with breaking down large molecules such as lipids or proteins. They perform an essential role in helping cells dispose of obsolete or harmful components; in fact, defects in lysosome function are associated with a range of health conditions. For instance, many genes associated with an increased risk of developing Parkinson's disease code for proteins required for lysosomes to work properly, such as the kinase LRRK2. Previous work has shown that this enzyme gets recruited to the surface of damaged lysosomes, where it can modulate the function of another set of molecular actors by modifying them through a chemical process known as phosphorylation. Such activity is increased in harmful versions of LRRK2 linked to Parkinson's disease. However, the molecular mechanisms which control LRRK2 activity or its recruitment to lysosomes remain unclear. To examine this question, Wang, Bondar et al. first performed a targeted screen to identify proteins that can regulate LRRK2 activity. This revealed that Rab12, one of molecular actors that LRRK2 phosphorylates, can in turn modulate the activity of the enzyme. Further imaging and biochemical experiments then showed that Rab12 is recruited to damaged lysosomes and that this step was in fact necessary for LRRK2 to also relocate to these compartments. The data suggest that this Rab12-driven recruitment process increases the local concentration of LRRK2 near its Rab targets on the membrane of damaged lysosomes, and therefore leads to enhanced LRRK2 activity. Crucially, Wang, Bondar et al. showed that Rab12 also plays a role in the increased LRRK2 activity observed with two Parkinson's disease-linked mutations (one in LRRK2 itself and one in another lysosomal regulator, VPS35), suggesting that increased LRRK2 concentration on lysosomes may be a conserved mechanism that leads to increased LRRK2 activity in disease. Overall, these results highlight a new, Rab12-dependent mechanism that results in enhanced activity at the lysosomal membrane with variants associated with Parkinson's disease, and for LRRK2 in general when lysosomes are damaged. This knowledge will be helpful to develop therapeutic strategies that target LRRK2, and to better understand how increased LRRK2 activity and lysosomal injury may be linked to Parkinson's disease.

Failure to Thrive: Impaired BDNF Transport along the Cortical-Striatal Axis in Mouse Q140 Neurons of Huntington's Disease.

Boosting trophic support to striatal neurons by increasing levels of brain-derived neurotrophic factor (BDNF) has been considered as a target for therapeutic intervention for several neurodegenerative diseases, including Huntington's disease (HD). To aid in the implementation of such a strategy, a thorough understanding of BDNF cortical-striatal transport is critical to help guide its strategic delivery. In this manuscript, we investigate the dynamic behavior of BDNF transport along the cortical-striatal axis in Q140 primary neurons, a mouse model for HD. We examine this by using single-molecule labeling of BDNF conjugated with quantum dots (QD-BDNF) to follow the transport along the cortical-striatal axis in a microfluidic chamber system specifically designed for the co-culture of cortical and striatal primary neurons. Using this approach, we observe a defect of QD-BDNF transport in Q140 neurons. Our study demonstrates that QD-BDNF transport along the cortical-striatal axis involves the impairment of anterograde transport within axons of cortical neurons, and of retrograde transport within dendrites of striatal neurons. One prominent feature we observe is the extended pause time of QD-BDNF retrograde transport within Q140 striatal dendrites. Taken together, these finding support the hypothesis that delinquent spatiotemporal trophic support of BDNF to striatal neurons, driven by impaired transport, may contribute to the pathogenesis of HD, providing us with insight into how a BDNF supplementation therapeutic strategy may best be applied for HD.

Preclinical and clinical evaluation of the LRRK2 inhibitor DNL201 for Parkinson's disease.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic risk factors for Parkinson's disease (PD). Increased LRRK2 kinase activity is thought to impair lysosomal function and may contribute to the pathogenesis of PD. Thus, inhibition of LRRK2 is a potential disease-modifying therapeutic strategy for PD. DNL201 is an investigational, first-in-class, CNS-penetrant, selective, ATP-competitive, small-molecule LRRK2 kinase inhibitor. In preclinical models, DNL201 inhibited LRRK2 kinase activity as evidenced by reduced phosphorylation of both LRRK2 at serine-935 (pS935) and Rab10 at threonine-73 (pT73), a direct substrate of LRRK2. Inhibition of LRRK2 by DNL201 demonstrated improved lysosomal function in cellular models of disease, including primary mouse astrocytes and fibroblasts from patients with Gaucher disease. Chronic administration of DNL201 to cynomolgus macaques at pharmacologically relevant doses was not associated with adverse findings. In phase 1 and phase 1b clinical trials in 122 healthy volunteers and in 28 patients with PD, respectively, DNL201 at single and multiple doses inhibited LRRK2 and was well tolerated at doses demonstrating LRRK2 pathway engagement and alteration of downstream lysosomal biomarkers. Robust cerebrospinal fluid penetration of DNL201 was observed in both healthy volunteers and patients with PD. These data support the hypothesis that LRRK2 inhibition has the potential to correct lysosomal dysfunction in patients with PD at doses that are generally safe and well tolerated, warranting further clinical development of LRRK2 inhibitors as a therapeutic modality for PD.

Understanding LRRK2 kinase activity in preclinical models and human subjects through quantitative analysis of LRRK2 and pT73 Rab10.

Variants in the leucine-rich repeat kinase 2 (LRRK2) gene are associated with increased risk for familial and sporadic Parkinson's disease (PD). Pathogenic variants in LRRK2, including the common variant G2019S, result in increased LRRK2 kinase activity, supporting the therapeutic potential of LRRK2 kinase inhibitors for PD. To better understand the role of LRRK2 in disease and to support the clinical development of LRRK2 inhibitors, quantitative and high-throughput assays to measure LRRK2 levels and activity are needed. We developed and applied such assays to measure the levels of LRRK2 as well as the phosphorylation of LRRK2 itself or one of its substrates, Rab10 (pT73 Rab10). We observed increased LRRK2 activity in various cellular models of disease, including iPSC-derived microglia, as well as in human subjects carrying the disease-linked variant LRRK2 G2019S. Capitalizing on the high-throughput and sensitive nature of these assays, we detected a significant reduction in LRRK2 activity in subjects carrying missense variants in LRRK2 associated with reduced disease risk. Finally, we optimized these assays to enable analysis of LRRK2 activity following inhibition in human peripheral blood mononuclear cells (PBMCs) and whole blood, demonstrating their potential utility as biomarkers to assess changes in LRRK2 expression and activity in the clinic.

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.

Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System.

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of their normal in vivo interactions. Slices obtained from a variety of transgenic mouse lines or use of viral vectors for expression of fluorescently tagged proteins or reporters in wild type brain slices allow for high-resolution imaging by fluorescence microscopy. Although several methods have been developed for imaging brain slices, combining slice culture with the ability to perform repetitive high-resolution imaging of specific cells in live slices over long time periods has posed problems. This is especially true when viral vectors are used for expression of exogenous proteins since this is best done in a closed system to protect users and prevent cross contamination. Simple modifications made to the roller tube brain slice culture method that allow for repetitive high-resolution imaging of slices over many weeks in an enclosed system are reported. Culturing slices on photoetched coverslips permits the use of fiducial marks to rapidly and precisely reposition the stage to image the identical field over time before and after different treatments. Examples are shown for the use of this method combined with specific neuronal staining and expression to observe changes in hippocampal slice architecture, viral-mediated neuronal expression of fluorescent proteins, and the development of cofilin pathology, which was previously observed in the hippocampus of Alzheimer's disease (AD) in response to slice treatment with oligomers of amyloid-ß (Aß) peptide.

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.

A SxIP motif interaction at the microtubule plus end is important for processive retrograde axonal transport.

The retrograde transport of endosomes within axons proceeds with remarkable uniformity despite having to navigate a discontinuous microtubule network. The mechanisms through which this navigation is achieved remain elusive. In this report, we demonstrate that access of SxIP motif proteins, such as BPAG1n4, to the microtubule plus end is important for the maintenance of processive and sustained retrograde transport along the axon. Disruption of this interaction at the microtubule plus end significantly increases endosome stalling. Our study thus provides strong insight into the role of plus-end-binding proteins in the processive navigation of cargo within the axon.

AxonQuant: A Microfluidic Chamber Culture-Coupled Algorithm That Allows High-Throughput Quantification of Axonal Damage.

Published methods for imaging and quantitatively analyzing morphological changes in neuronal axons have serious limitations because of their small sample sizes, and their time-consuming and nonobjective nature. Here we present an improved microfluidic chamber design suitable for fast and high-throughput imaging of neuronal axons. We developed the AxonQuant algorithm, which is suitable for automatic processing of axonal imaging data. This microfluidic chamber-coupled algorithm allows calculation of an 'axonal continuity index' that quantitatively measures axonal health status in a manner independent of neuronal or axonal density. This method allows quantitative analysis of axonal morphology in an automatic and nonbiased manner. Our method will facilitate large-scale high-throughput screening for genes or therapeutic compounds for neurodegenerative diseases involving axonal damage. When combined with imaging technologies utilizing different gene markers, this method will provide new insights into the mechanistic basis for axon degeneration. Our microfluidic chamber culture-coupled AxonQuant algorithm will be widely useful for studying axonal biology and neurodegenerative disorders.

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.

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.

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.

Mechanisms of neuronal growth cone guidance: an historical perspective.

At the distal most aspect of motile extending axons and dendrites lies the growth cone, a hand like macroorganelle of membrane bound cytoskeleton, packed with receptors, adhesion molecules, molecular motors, and an army of regulatory and signaling proteins. Splayed out along the substratum in vitro, the growth cone resembles an open hand with bundles of filamentous actin, barbed ends outstretched, as if fingers extending from a central domain of dynamic microtubule plus ends. The growth cone acts first as a sensory platform, analyzing the environment ahead for the presence of guidance cues, secondly as a mechanical dynamo establishing focal contact with the extracellular matrix to drive processive forward outgrowth, and thirdly as a forward biochemical command center where signals are interrogated to inform turning, extension, retraction, or branching. During his career, Paul Letourneau has made major contributions to our understanding of how growth cones respond to their environment. Here, we will summarize some of these major advances in their historical context. Letourneau's contributions have provided insights into cytoskeletal organization, growth cone dynamics, and signaling pathways. His recent work has described some important molecules and molecular mechanisms involved in growth cone turning. Although much remains to be understood about this important and intriguing structure, Letourneau's contributions have provided us with "growth cone guidance."

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.

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

BACKGROUND: Previously 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. RESULTS: Here we demonstrate that a gel filtration fraction of 7PA2 cell-secreted SDS-stable human Aß dimers and trimers (Aßd/t) induces maximal neuronal rod response at ~250 pM. This is 4,000-fold more active than traditionally prepared human Aß oligomers, which contain SDS-stable trimers and tetramers, but are devoid of dimers. When incubated under tyrosine oxidizing conditions, synthetic human but not rodent Aß1-42, 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 Aßd/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 Aß monomers are not active, suggesting oxidized SDS-stable Aß1-42 dimers in a low-n state are the most active rod-inducing species. Aßd/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 Aßd/t washout. Overexpression of cofilin phosphatases increase rod formation when expressed alone and exacerbate rod formation when coupled with Aßd/t, whereas overexpression of a cofilin kinase inhibits Aßd/t-induced rod formation. CONCLUSIONS: Together these data support a mechanism by which Aßd/t alters the actin cytoskeleton via effects on cofilin in neurons critical to learning and memory.

Actin and Diseases of the Nervous System.

Abnormal regulation of the actin cytoskeleton results in several pathological conditions affecting primarily the nervous system. Those of genetic origin arise during development, but others manifest later in life. Actin regulation is also affected profoundly by environmental factors that can have sustained consequences for the nervous system. Those consequences follow from the fact that the actin cytoskeleton is essential for a multitude of cell biological functions ranging from neuronal migration in cortical development and dendritic spine formation to NMDA receptor activity in learning and alcoholism. Improper regulation of actin, causing aggregation, can contribute to the neurodegeneration of amyloidopathies, such as Down's syndrome and Alzheimer's disease. Much progress has been made in understanding the molecular basis of these diseases.

The outcome from legalizing Sunday packaged alcohol sales on traffic accidents in New Mexico.

We attempt to disentangle the effect on alcohol-related accidents and fatal crashes when New Mexico lifted its ban on Sunday packaged alcohol sales on July 1, 1995. Using crash incidents between January 1990 and December 2005, from data maintained by the Division of Government Research in New Mexico, we estimate a negative binomial model that controls for unobservable factors affecting overall accidents. One of these factors is an increase in New Mexico's speed limits in 1996. We find no statistically significant increase in total alcohol-related accidents or alcohol-related fatal crashes on Sundays after the repeal of the ban.

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.

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-beta (Abeta) 1-42 peptide, undergoes dephosphorylation (activation) and forms rod-shaped actin bundles (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 Abeta1-40 at high concentrations, and these neurons undergo dystrophic morphological changes, including accumulation of pretangle phosphorylated-tau. Therefore, extremes in phosphoregulation of cofilin by different forms of Abeta may explain much of the Alzheimer's disease pathology and provide mechanisms for synaptic loss and plaque expansion.

Beta-secretase-cleaved amyloid precursor protein accumulates at actin inclusions induced in neurons by stress or amyloid beta: 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 Alzheimer's disease (AD) brain (Minamide et al., 2000). Here, we show rods are found in brains from transgenic AD mice. Soluble forms of amyloid beta (Abeta(1-42)) 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 Abeta(1-42). Abeta(1-42) induces the activation (dephosphorylation) of ADF/cofilin in neurons that form rods. Vesicles containing amyloid precursor protein (APP), beta-amyloid cleavage enzyme, and presenilin-1, a component of the gamma-secretase complex, accumulate at rods. The beta-secretase-cleaved APP (either beta-C-terminal fragment of APP or Abeta) also accumulates at rods. These results suggest that rods, formed in response to either Abeta or some other stress, block the transport of APP and enzymes involved in its processing to Abeta. These stalled vesicles may provide a site for producing Abeta(1-42), which may in turn induce more rods in surrounding neurons, and expand the degenerative zone resulting in plaque formation.