Formation Of Dystrophic Neurites Research Articles

Transcriptome and proteome profiling reveals TREM2-dependent and -independent glial response and metabolic perturbation in an Alzheimer’s mouse model

Elucidating the intricate molecular mechanisms of Alzheimer's disease (AD) requires a multidimensional analysis incorporating various omics data. In this study, we employed transcriptome and proteome profiling of AppNL-G-F, a human APP knock-in model of amyloidosis, at the early and mid-stages of amyloid-beta (Aβ) pathology to delineate the impacts of Aβ deposition on brain cells. By contrasting AppNL-G-F mice with TREM2 (Triggering receptor expressed on myeloid cells 2) knockout models, our study further investigates the role of TREM2, a well-known AD risk gene, in influencing microglial responses to Aβ pathology. Our results highlight altered microglial states as a central feature of Aβ pathology, characterized by the significant upregulation of microglia-specific genes related to immune responses such as complement system and antigen presentation, and catabolic pathways such as phagosome formation and lysosome biogenesis. The absence of TREM2 markedly diminishes the induction of these genes, impairs Aβ clearance, and exacerbates dystrophic neurite formation. Importantly, TREM2 is required for the microglial engagement with Aβ plaques and the formation of compact Aβ plaque cores. Furthermore, this study reveals substantial disruptions in energy metabolism and protein synthesis, signaling a shift from anabolism to catabolism in response to Aβ deposition. This metabolic alteration, coupled with a decrease in synaptic protein abundance, occurs independently of TREM2, suggesting the direct effects of Aβ deposition on synaptic integrity and plasticity. In summary, our findings demonstrate altered microglial states and metabolic disruption following Aβ deposition, offering mechanistic insights into Aβ pathology and highlighting the potential of targeting these pathways in AD therapy.

Most dystrophic neurites in the common 5xFAD Alzheimer mouse model originate from axon terminals

How dystrophic neurites form around amyloid plaques is a key aspect of understanding the early pathophysiology of Alzheimer's disease. At present, three hypotheses prevail: (1) dystrophies result from extracellular amyloid-beta (Aβ) toxicity; (2) dystrophies results from accumulation of Aβ into distal neurites; and (3) dystrophies represent blebbing of the somatic membrane of a neuron with high Aβ load. We utilized a unique feature of the common 5xFAD AD mouse model to test these hypotheses. Cortical layer 5 pyramidal neurons show intracellular APP and Aβ accumulation before amyloid plaque formation while dentate granule cells in these mice show no APP accumulation at any age. However, the dentate gyrus shows amyloid plaques by 3 months of age. By a careful confocal microscopic analysis we found no evidence of severe degeneration in amyloid laden layer 5 pyramidal neurons in contrast to hypothesis 3. Using injecting red fluorescent marker into lateral entorhinal projection neurons in 5xFAD mice with endogenous green fluorescent protein (GFP) in dentate granule cells we could demonstrate that all dystrophies is outer molecular layer originate from the axon terminal of entorhinal projection neurons. Immunostaining with vesicular glutamate transporter supported the axonal nature of the dystrophies in the acellular dentate molecular layer. We observed few small dystrophies in the GFP labeled granule cell dendrites. In general GFP labeled dendrites appear normal around the amyloid plaques. These findings favor hypothesis 2 as the most likely mechanism of dystrophic neurite formation.

Definition of the contribution of an Osteopontin-producing CD11c+ microglial subset to Alzheimer’s disease

Alzheimer's disease (AD) is the most common form of incurable dementia and represents a critical public health issue as the world's population ages. Although microglial dysregulation is a cardinal feature of AD, the extensive heterogeneity of these immunological cells in the brain has impeded our understanding of their contribution to this disease. Here, we identify a pathogenic microglial subset which expresses the CD11c surface marker as the sole producer of Osteopontin (OPN) in the 5XFAD mouse model of AD. OPN production divides Disease-Associated Microglia (DAM) into two functionally distinct subsets, i.e., a protective CD11c+OPN- subset that robustly ingests amyloid β (Aβ) in a noninflammatory fashion and a pathogenic CD11c+OPN+ subset that produces proinflammatory cytokines and fails to ingest significant amounts of Aβ. Genetic ablation of OPN or administration of monoclonal anti-OPN antibody to 5XFAD mice reduces proinflammatory microglia, plaque formation, and numbers of dystrophic neurites and results in improved cognitive function. Analysis of brain tissue from AD patients indicates that levels of OPN-producing CD11c+ microglia correlate strongly with the degree of cognitive deficit and AD neuropathology. These findings define an OPN-dependent pathway to disease driven by a distinct microglial subset, and identify OPN as a novel therapeutic target for potentially effective immunotherapy to treat AD.

Poloxamer-188 Exacerbates Brain Amyloidosis, Presynaptic Dystrophies, and Pathogenic Microglial Activation in 5XFAD Mice.

Alzheimer's disease (AD) is initiated by aberrant accumulation of amyloid beta (Aβ) protein in the brain parenchyma. The microenvironment surrounding amyloid plaques is characterized by the swelling of presynaptic terminals (dystrophic neurites) associated with lysosomal dysfunction, microtubule disruption, and impaired axonal transport. Aβ-induced plasma membrane damage and calcium influx could be potential mechanisms underlying dystrophic neurite formation. We tested whether promoting membrane integrity by brain administration of a safe FDA approved surfactant molecule poloxamer-188 (P188) could attenuate AD pathology in vivo. Three-month-old 5XFAD male mice were administered several concentrations of P188 in the brain for 42 days with mini-osmotic pumps. After 42 days, mice were euthanized and assessed for amyloid pathology, dystrophic neurites, pathogenic microglia activation, tau phosphorylation, and lysosomal / vesicular trafficking markers in the brain. P188 was lethal at the highest concentration of 10mM. Lower concentrations of P188 (1.2, 12, and 120μM) were well tolerated. P188 increased brain Aβ burden, potentially through activation of the γ-secretase pathway. Dystrophic neurite pathology was exacerbated in P188 treated mice as indicated by increased LAMP1 accumulation around Aβ deposits. Pathogenic microglial activation was increased by P188. Total tau levels were decreased by P188. Lysosomal enzyme cathepsin D and calciumdependent vesicular trafficking regulator synaptotagmin-7 (SYT7) were dysregulated upon P188 administration. P188 brain delivery exacerbated amyloid pathology, dystrophic neurites, and pathogenic microglial activation in 5XFAD mice. These effects correlated with lysosomal dysfunction and dysregulation of plasma membrane vesicular trafficking. P188 is not a promising therapeutic strategy against AD pathogenesis.

Oral nimodipine treatment has no effect on amyloid pathology or neuritic dystrophy in the 5XFAD mouse model of amyloidosis.

Dysregulation of calcium homeostasis has been hypothesized to play a role in Alzheimer’s disease (AD) pathogenesis. Increased calcium levels can impair axonal transport, disrupt synaptic transmission, and ultimately lead to cell death. Given the potential role of calcium dyshomeostasis in AD, there is interest in testing the ability of already approved drugs targeting various calcium channels to affect amyloid pathology and other aspects of disease. The objective of this study was to test the effects of FDA-approved L-type calcium channel antagonist nimodipine on amyloid accumulation and dystrophic neurite formation in 5XFAD mice, a mouse model of amyloid pathology. 5XFAD transgenic mice and non-transgenic littermates were treated with vehicle or nimodipine-containing chow from two to eight months of age, then brains were harvested and amyloid pathology assessed by immunoblot and immunofluorescence microscopy analyses. Nimodipine was well tolerated and crossed the blood brain barrier, as expected, but there was no effect on Aβ accumulation or on the relative amount of neuritic dystrophy, as assessed by either immunoblot, dot blot or immunofluorescence imaging of Aβ42 and dystrophic neurite marker LAMP1. While we conclude that nimodipine treatment is not likely to improve amyloid pathology or decrease neuritic dystrophy in AD, it is worth noting that nimodipine did not worsen the phenotype suggesting its use is safe in AD patients.

Dynactin 6 deficiency enhances aging-associated dystrophic neurite formation in mouse brains

Formation of Reticulon 3 (RTN3)-immunoreactive dystrophic neurites (RIDNs) occurs early during the growth of amyloid plaques in Alzheimer's disease (AD) brains. We have shown that RIDNs in AD and aging mouse brains are composed of abnormally clustered tubular endoplasmic reticulum (ER) and degenerating mitochondria. To understand RTN3-mediated abnormal tubular ER clustering, we aimed to identify proteins that interact with RTN3 and impact accumulation of tubular ER in RIDNs. We found that the N-terminal domain of RTN3, which is unique among RTN family members, specifically interacted with dynactin 6 (DCTN6), a protein involved in dynein-mediated retrograde transport of cargo vesicles. DCTN6 protein levels decrease with aging in the hippocampal regions of WT mice. We found that DCTN6 deficiency enhanced RTN3 protein levels, high molecular weight RTN3 levels, and hippocampus-specific RIDN formation in aging brains of transgenic mice overexpressing RTN3. Our results suggest that the DCTN6-RTN3 interaction mediates tubular ER trafficking in axons, and a DCTN6 deficiency in the hippocampus impairs axonal ER trafficking, leading to abnormal ER accumulation and RIDN formation in brains of aging mice.

CSF1R inhibitor abrogates tau propagation exacerbated in APPNL‐G‐F knock‐in mice but enhances fibrillar beta‐amyloidosis and dystrophic neurite formation in the brain

AbstractBackgroundMicroglia are the primary innate immune cells in the brain. They can phagocytose apoptotic neurons and dystrophic neurites containing aggregated and phosphorylated tau (p‐tau) in tauopathies, and possibly spread pathological tau via extracellular vesicles (EVs) release. Proteinopathic stress like amyloid plaque accumulation can transform microglia into a neurodegenerative phenotype (MGnD), possessing enhanced phagocytic and exocytotic functions. Thus, MGnD could exacerbate tau spreading throughout the brain. To determine the role of MGnD on tau propagation, we employed microglia depletion in adeno‐associated virus (AAV)‐mediated tau propagation mouse models.MethodC57BL/6 (WT) and APPNL‐G‐F mice were fed with a CSF1R inhibitor (PLX5622) or control chow for one month before and after stereotaxic injections of AAV2/6‐SYN1‐P301Ltau expressing P301L tau mutant into the medial entorhinal cortex (MEC) at 5 months of age. Propagation of tau to the dentate granular cells of the hippocampus, amyloid plaque formation, and association of microglia and p‐tau with plaques were assessed by immunohistochemistry after one month. MGnD and homeostatic microglia were separately isolated from APPNL‐G‐F mouse brains by FACS and evaluated for expression of EV marker molecules.ResultFACS data showed higher percentage of Clec7a (MGnD marker)‐positive microglia (20.8%) in APPNL‐G‐F compared to WT. Immunohistochemistry of APPNL‐G‐F mouse brains revealed strong positivity of Clec7a+ microglia surrounding both amyloid plaques and plaque‐associated p‐tau. APPNL‐G‐F mice exhibited approximately a 10‐fold increase in tau propagation compared to WT mice. Strikingly, PLX5622 treatment, which depleted ∼ 99% of microglia, showed 74 and 87% reduction of tau propagation in WT and APPNL‐G‐F groups, respectively. Contrarily, PLX5622 treatment increased intensity of plaque associated p‐tau along with increased size and number of amyloid plaques in the APPNL‐G‐F mice, suggesting their regulation by MGnD. Gene expression of EV markers, CD9 and CD63, was upregulated in Clec7a+ microglia compared to Clec7– microglia isolated from APPNL‐G‐F mice, suggesting enhanced microglial EV synthesis. This was further supported by the significant colocalization of Tsg101, an exosome marker, in Clec7a+ MGnD.ConclusionTau propagation may be influenced by engulfment of pathological tau seeds by microglia and secretion through EVs, which is exacerbated in MGnD in APPNL‐G‐F mice and mitigated by PLX5622 treatment.

Dynactin 6 deficiency enhances tubular ER‐associated dystrophic neurites formation

AbstractBackgroundThe formation of dystrophic neurites (DNs) is a contributing factor to synaptic dysfunction in Alzheimer’s disease (AD) patients’ brains. We have shown that tubular endoplasmic reticulum (ER) shaping proteins reticulon 3 (RTN3), REEP2 and REEP5 are participated in the formation DNs, named as RTN3 immunoreactive DNs or RIDNs. RIDNs are appeared as clustered or dispersed forms in AD and aging mouse brain, respectively. Ultrastructural investigation showed that RIDNs are constitute with abnormal clustering of tubular ER. The over expression of RTN3 accelerates aging associated disperse RIDNs formation in Tg‐RTN3 mouse brain hippocampus. To determine a molecule that may cause RTN3 mediated abnormal tubular ER clustering, we aimed to identify RTN3 interacting proteins and to elucidate its functional roles in tabular ER accumulation and RIDNs formation in aging and AD brain.MethodsYeast two‐hybrid system, co‐immunoprecipitation, domain mapping and immune‐confocal studies to identify RTN3 interacting proteins. WB was performed to measure the protein level. Formation of RIDNs in mouse brains were detected using immuno‐confocal study. AAV9‐RTN3‐mCherry and AAV9‐DCTN‐GFP were injected at hippocampus of wild type mice to over expressed RTN3 and DCTN6.ResultsWe have identified that N‐terminal domain of RTN3 interacts with dynactin 6 (DCTN6), a protein component that involved in dynein‐mediated retrograde transport of cargo vesicles. Our immune‐confocal studies on DCTN6 and RTN3 over expressed cells and primary cultured neuron indicate that RTN3‐DCTN6 interaction likely mediates the tubular ER trafficking. The DCTN6 protein level decreases during aging and it reduces in hippocampus compared to cortical samples of the same age mice. To understand the functional roles of DCTN6 in RIDNs formation during aging and beta‐amyloid plaque deposition, DCTN6 deficient Tg‐RTN3 and 5xFAD mice were generated. Tg‐RTN3; DCTN6+/‐ showed reduced DCTN6 protein level and enhanced hippocampus specific RINDs formation compare to Tg‐RTN3; DCTN6+/+ mouse. We are investigating whether RIDNs formation are increases in DCTN6 deficient 5xFAD mice and whether AAV9‐DCTN6 overexpression reduces RIDNs formation in AAV9‐RTN3 overexpressed mouse brain.ConclusionRTN3‐DCTN6 interaction likely mediates the tubular ER transport in axons and the DCTN6 deficiency is a contributing factor for tubular ER accumulation, RIDNs formation in AD and aging brain.

Anti-A\u03b2 antibodies bound to neuritic plaques enhance microglia activity and mitigate tau pathology

The brain pathology of Alzheimer’s disease (AD) is characterized by the misfolding and aggregation of both the amyloid beta (Aβ) peptide and hyperphosphorylated forms of the tau protein. Initial Aβ deposition is considered to trigger a sequence of deleterious events contributing to tau pathology, neuroinflammation and ultimately causing the loss of synapses and neurons. To assess the effect of anti-Aβ immunization in this context, we generated a mouse model by overexpressing the human tau protein in the hippocampus of 5xFAD mice. Aβ plaque deposition combined with human tau overexpression leads to an array of pathological manifestations including the formation of tau-positive dystrophic neurites and accumulation of hyperphosphorylated tau at the level of neuritic plaques. Remarkably, the presence of human tau reduces microglial clustering in proximity to the Aβ plaques, which may affect the barrier role of microglia. In this mouse model, continuous administration of anti-Aβ antibodies enhances the clustering of microglial cells even in the presence of tau. Anti-Aβ immunization increases plaque compaction, reduces the spread of tau in the hippocampal formation and prevents the formation of tau-positive dystrophic neurites. However, the treatment does not significantly reduce tau-induced neurodegeneration in the dentate gyrus. These results highlight that anti-Aβ immunization is able to enhance microglial activity around neuritic plaques, mitigating part of the tau-induced pathological manifestations.

Clustering of activated microglia occurs before the formation of dystrophic neurites in the evolution of Aβ plaques in Alzheimer's disease.

Alzheimer's disease (AD) is a late-onset disease that has proved difficult to model. Microglia are implicated in AD, but reports vary on precisely when and how in the sequence of pathological changes they become involved. Here, post-mortem human tissue from two differentially affected regions of the AD brain and from non-demented individuals with a high load of AD-type pathology (high pathology controls) was used to model the disease time course in order to determine how microglial activation relates temporally to the deposition of hallmark amyloid-β (Aβ) and hyperphosphorylated microtubule associated protein tau pathology. Immunofluorescence against the pan-microglial marker, ionised calcium-binding adapter molecule 1 (IBA1), Aβ and tau, was performed in the primary motor cortex (PMC), a region relatively spared of AD pathological changes, and compared to the severely affected inferior temporal cortex (ITC) in the same cases. Unlike the ITC, the PMC in the AD cases was spared of any degenerative changes in cortical thickness and the density of Betz cells and total neurons. The clustering of activated microglia was greatest in the PMC of AD cases and high pathology controls compared to the ITC. This suggests microglial activation is most prominent in the early phases of AD pathophysiology. Nascent tau inclusions were found in neuritic plaques in the PMC but were more numerous in the ITC of the same case. This shows that tau positive neuritic plaques begin early in AD which is likely of pathogenic importance, however major tau deposition follows the accumulation of Aβ and clustering of activated microglia. Importantly, findings presented here demonstrate that different states of microglial activation, corresponding to regional accumulations of Aβ and tau, are present simultaneously in the same individual; an important factor for consideration if targeting these cells for therapeutic intervention.

Short-term fish oil supplementation applied in presymptomatic stage of Alzheimer's disease enhances microglial/macrophage barrier and prevents neuritic dystrophy in parietal cortex of 5xFAD mouse model.

Dystrophic neurites and activated microglia are one of the main neuropathological characteristics of Alzheimer's disease (AD). Although the use of supplements with omega-3 fatty acids has been associated with reduced risk and lessened AD pathology, it still remains elusive whether such a treatment could affect dystrophic neurites (DNs) formation and microglia/macrophage behavior in the early phase of disease. We analyzed the effects of short-term (3 weeks) fish oil supplementation on DNs formation, tau hyperphosphorylation, Amyloid-beta peptide 1–42 (Aβ42) levels and microglial/macrophage response to AD pathology in the parietal cortex of 4-month-old 5xFAD mice, a mouse model of AD. The present study shows for the first time that short-term FO supplementation applied in presymptomatic stage of AD, alters the behaviour of microglia/macrophages prompting them to establish a physical barrier around amyloid plaques. This barrier significantly suppresses DNs formation through the reduction of both Aβ content and tau hyperphosphorylation. Moreover, the short-term FO treatment neither suppresses inflammation nor enhances phagocytic properties of microglia/macrophages in the response to Aβ pathology, the effects most commonly attributed to the fish oil supplementation. Our findings suggest that fish oil consumption may play an important role in modulating microglial/macrophage response and ameliorating the AD pathology in presymptomatic stage of Alzheimer's disease.

Formation of Dystrophic Neurites in Alzheimer's Disease

Dystrophic neurites (DNs) are abnormal neuronal processes characterized microscopically by aberrant sprouting, dystrophic expansion, and accumulation of various cellular organelles and cytoskeletal/signaling proteins. DNs occur around neuritic plaques in the brains of subjects with Alzheimer's disease (AD) and various other neurological conditions. Despite decades of research, the mechanism underlying the development of DNs remains unclear. This talk will discuss the understanding of DNs in historic perspectives, with a focus on the relationship between the formation of DNs and beta‐amyloid deposition according to recent studies using postmortem human brains and AD animal models.Support or Funding InformationAAA Symposium: Progress of Human Brain Banking in China: Construction and Research. Supported by AAA (American Association of Anatomists) and CSAS (Chinese Society for Anatomical Sciences).

Presynaptic dystrophic neurites surrounding amyloid plaques are sites of microtubule disruption, BACE1 elevation, and increased Aβ generation in Alzheimer’s disease

Alzheimer’s disease (AD) is characterized by amyloid plaques composed of the β-amyloid (Aβ) peptide surrounded by swollen presynaptic dystrophic neurites consisting of dysfunctional axons and terminals that accumulate the β-site amyloid precursor protein (APP) cleaving enzyme (BACE1) required for Aβ generation. The cellular and molecular mechanisms that govern presynaptic dystrophic neurite formation are unclear, and elucidating these processes may lead to novel AD therapeutic strategies. Previous studies suggest Aβ may disrupt microtubules, which we hypothesize have a critical role in the development of presynaptic dystrophies. To investigate this further, here we have assessed the effects of Aβ, particularly neurotoxic Aβ42, on microtubules during the formation of presynaptic dystrophic neurites in vitro and in vivo. Live-cell imaging of primary neurons revealed that exposure to Aβ42 oligomers caused varicose and beaded neurites with extensive microtubule disruption, and inhibited anterograde and retrograde trafficking. In brain sections from AD patients and the 5XFAD transgenic mouse model of amyloid pathology, dystrophic neurite halos with BACE1 elevation around amyloid plaques exhibited aberrant tubulin accumulations or voids. At the ultrastructural level, peri-plaque dystrophies were strikingly devoid of microtubules and replete with multi-lamellar vesicles resembling autophagic intermediates. Proteins of the microtubule motors, kinesin and dynein, and other neuronal proteins were aberrantly localized in peri-plaque dystrophies. Inactive pro-cathepsin D also accumulated in peri-plaque dystrophies, indicating reduced lysosomal function. Most importantly, BACE1 accumulation in peri-plaque dystrophies caused increased BACE1 cleavage of APP and Aβ generation. Our study supports the hypothesis that Aβ induces microtubule disruption in presynaptic dystrophic neurites that surround plaques, thus impairing axonal transport and leading to accumulation of BACE1 and exacerbation of amyloid pathology in AD.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-016-1558-9) contains supplementary material, which is available to authorized users.

The role of calsyntenin-3 in dystrophic neurite formation in Alzheimer's disease brain.

β-Amyloid (Aβ) oligomers may play an important role in the early pathogenesis of Alzheimer's disease: cognitive impairment caused by synaptic dysfunction. Dystrophic neurites surrounding Aβ plaques, another pathological feature of Alzheimer's disease, are plaque-associated neuritic alterations preceding the appearance of synaptic loss. In the present review, we focus on the mechanism of dystrophic neurite formation by Aß oligomers, and discuss the neurotoxic role of Aβ-induced calsyntenin-3 in mediating dystrophic neurite formation.

Dysfunctional tubular endoplasmic reticulum constitutes a pathological feature of Alzheimer's disease.

Pathological features in Alzheimer’s brains include mitochondrial dysfunction and dystrophic neurites (DNs) in areas surrounding amyloid plaques. Using a mouse model that overexpresses reticulon 3 (RTN3) and spontaneously develops age-dependent hippocampal DNs, here we report that DNs contain both RTN3 and REEPs, topologically similar proteins that can shape tubular endoplasmic reticulum (ER). Importantly, ultrastructural examinations of such DNs revealed gradual accumulation of tubular ER in axonal termini, and such abnormal tubular ER inclusion is found in areas surrounding amyloid plaques in biopsy samples from AD brains. Functionally, abnormally clustered tubular ER induces enhanced mitochondrial fission in the early stages of DN formation and eventual mitochondrial degeneration at later stages. Furthermore, such DNs are abrogated when RTN3 is ablated in aging and AD mouse models. Hence, abnormally clustered tubular ER can be pathogenic in brain regions: disrupting mitochondrial integrity, inducing DNs formation and impairing cognitive function in AD and aging brains

Calsyntenin-3 C-Terminal Fragment Accumulates in Dystrophic Neurites Surrounding Aβ Plaques in Tg2576 Mouse and Alzheimer Disease Brains: Its Neurotoxic Role in Mediating Dystrophic Neurite Formation

Dystrophic neurites surrounding β-amyloid (Aβ) plaques precede neuronal death in Alzheimer disease. These neuritic alterations may be one of the initial stages for synaptic loss and dysfunction. However, intracellular pathways that cause local disruption of neuronal processes by Aβ remain to be fully elucidated. The identification of Aβ-induced genes that mediate neuritic pathology would provide considerable insight into the mechanisms of Alzheimer's disease. Previously, we reported that selective up-regulation of calsyntenin-3 (Cst-3) by Aβ and accumulation of neurotoxic Cst-3 in dystrophic neurites surrounding Aβ plaques may lead to local disruption of these neurites. Like amyloid precursor protein, Cst-3 undergoes two-step proteolytic processing: the primary cleavage with α-secretase generates an N-terminal ectodomain and a C-terminal fragment (CTF). The CTF is subsequently cleaved into p3 peptide and an intracellular domain via γ-secretase. It would be interesting to know whether accumulated Cst-3 in dystrophic neurites surrounding Aβ plaques is the full-length version or a CTF. Herein, we show that the CTF but not full-length Cst-3 accumulated in dystrophic neurites surrounding Aβ plaques in Tg2576 mouse and Alzheimer disease brains. Invitro experiments with Cst-3 fragments have revealed that only the CTF resulted in acceleration of neuronal death. These results indicate that accumulation of the neurotoxic CTF in neurites surrounding Aβ plaques may lead to local disruption of neuronal processes and development of dystrophic neurites.

Neurites containing the neurofilament-triplet proteins are selectively vulnerable to cytoskeletal pathology in Alzheimer’s disease and transgenic mouse models

Amyloid-β plaque accumulation in Alzheimer’s disease (AD) is associated with dystrophic neurite (DN) formation and synapse loss in principal neurons, but interneuron pathology is less clearly characterized. We compared the responses of neuronal processes immunoreactive for either neurofilament triplet (NF+) or calretinin (CR+) to fibrillar amyloid (Aβ) plaques in human end-stage and preclinical AD, as well as in APP/PS1 and Tg2576 transgenic mouse AD models. Neurites traversing the Aβ plaque core, edge, or periphery, defined as 50, 100, and 150% of the plaque diameter, respectively, in human AD and transgenic mouse tissue were compared to age-matched human and wild-type mouse controls. The proportion of NF+ neurites exhibiting dystrophic morphology (DN) was significantly larger than the proportion of dystrophic CR+ neurites in both human AD and transgenic mice (p < 0.01). Additionally, the number of NF+, but not CR+, DNs, correlated with Aβ plaque size. We conclude that CR+ interneurons appear to be more resistant than NF+ neurons to AD-mediated cytoskeletal pathology.

Reticulon 3 aggregation and its role in the formation of dystrophic neurites

'Alzheimer’s disease (AD), the most common cause of dementia in the elderly, is characterized by the presence of Ab plaques, which results in a progressive neuronal degeneration and subsequent cognitive decline. Dendrites and axons in the region surrounding the plaques may become damaged and swollen, forming what are known as dystrophic neurites (DNs). However, the mechanisms by which DNs affect cognitive function in AD patients are not understood. Methods While characterizing the reticulon (RTN)/Nogo family of proteins, which have been shown to negatively regulate BACE1 activity, we found that RTN3, which is primarily neuronally expressed, accumulates in a distinct population of DNs called RTN3 Immunoreactive Dystrophic Neurites (RIDNs) in the brains of AD patients and in APP transgenic mice, an animal model for AD. More importantly, transgenic mice overexpressing RTN3 (Tg-RTN3) produce RIDNs in the hippocampus at a young age, while wild-type mice produce similar RIDNs only at older ages. Results The importance of RIDNs in relation to cognitive decline is further evidenced by the finding that Tg-RTN3 mice where RIDNs are present show impaired learning and spatial memory, reduced spine density and synaptic plasticity. Molecular characterization of RIDNs indicates that RIDNs contain RTN3 aggregates and inducible expression of RTN3 in a mouse model reveals that RTN3 aggregates are formed irreversibly. Conclusion Together, our results suggest that inhibition of RTN3 aggregation is a promising approach for improving cognitive dysfunction associated with Alzheimer’s pathology.

High-Content Chemical and RNAi Screens for Suppressors of Neurotoxicity in a Huntington's Disease Model

To identify Huntington's Disease therapeutics, we conducted high-content small molecule and RNAi suppressor screens using a Drosophila primary neural culture Huntingtin model. Drosophila primary neurons offer a sensitive readout for neurotoxicty, as their neurites develop dysmorphic features in the presence of mutant polyglutamine-expanded Huntingtin compared to nonpathogenic Huntingtin. By tracking the subcellular distribution of mRFP-tagged pathogenic Huntingtin and assaying neurite branch morphology via live-imaging, we identified suppressors that could reduce Huntingtin aggregation and/or prevent the formation of dystrophic neurites. The custom algorithms we used to quantify neurite morphologies in complex cultures provide a useful tool for future high-content screening approaches focused on neurodegenerative disease models. Compounds previously found to be effective aggregation inhibitors in mammalian systems were also effective in Drosophila primary cultures, suggesting translational capacity between these models. However, we did not observe a direct correlation between the ability of a compound or gene knockdown to suppress aggregate formation and its ability to rescue dysmorphic neurites. Only a subset of aggregation inhibitors could revert dysmorphic cellular profiles. We identified lkb1, an upstream kinase in the mTOR/Insulin pathway, and four novel drugs, Camptothecin, OH-Camptothecin, 18β-Glycyrrhetinic acid, and Carbenoxolone, that were strong suppressors of mutant Huntingtin-induced neurotoxicity. Huntingtin neurotoxicity suppressors identified through our screen also restored viability in an in vivo Drosophila Huntington's Disease model, making them attractive candidates for further therapeutic evaluation.

Calretinin Interneurons are Early Targets of Extracellular Amyloid-β Pathology in PS1/AβPP Alzheimer Mice Hippocampus

Specific neuronal networks are preferentially affected in the early stages of Alzheimer's disease (AD). The distinct subpopulations of hippocampal inhibitory GABAergic system have been shown to display differential vulnerability to neurodegeneration in AD. We have previously reported a substantial loss of SOM/NPY interneurons, whereas those expressing parvalbumin were unaltered, in the hippocampus of 6 month-old PS1/AbetaPP transgenic mice. In the present study, we now investigated the pathological changes of hippocampal calretinin (CR) interneurons in this PS1/AbetaPP model from 2 to 12 months of age. The total number of CR-immunoreactive inhibitory cells was determined by stereology in CA1 and CA2/3 subfields. Our findings show a substantial decrease (35%-45%) of CR-positive interneurons in both hippocampal subfields of PS1/AbetaPP mice at very early age (4 months) compared to age-matched control mice. This decrease was accompanied by a reduced CR mRNA content as determined by quantitative RT-PCR. However, the number of another hippocampal CR-positive population (belonging to Cajal-Retzius cells) was not affected. The selective early loss of CR-interneurons was parallel to the appearance of extracellular Abeta deposits, preferentially in CR-axonal fields, and the formation of dystrophic neurites. This specific GABAergic subpopulation plays a crucial role in the generation of synchronous rhythmic activity in hippocampus by controlling other interneurons. Therefore, early alterations of hippocampal inhibitory functionality in AD, caused by select CR-cells neurodegeneration, could result in cognitive impairments seen in initial stages of the disease.