Inhibitory Roles of Apolipoprotein E Christchurch Astrocytes in Curbing Tau Propagation Using Human Pluripotent Stem Cell-Derived Models.

Genetic variants in the apolipoprotein E (APOE) gene affect the onset and progression of Alzheimer's disease (AD). The APOE Christchurch (APOE Ch) variant has been identified as the most prominent candidate for preventing the onset and progression of AD. In this study, we generated isogenic APOE3Ch/3Ch human-induced pluripotent stem cells (iPSCs) from APOE3/3 healthy control female iPSCs and induced them into astrocytes. RNA expression analysis revealed the inherent resilience of APOE3Ch/3Ch astrocytes to induce a reactive state in response to inflammatory cytokines. Moreover, cytokine treatment changed astrocytic morphology with more complexity in APOE3/3 astrocytes, but not in APOE3Ch/3Ch astrocytes, indicating resilience of the rare variant to a reactive state. Interestingly, we observed robust morphological alterations containing more intricate processes when cocultured with iPSC-derived cortical neurons, in which APOE3Ch/3Ch astrocytes reduced complexity compared with APOE3/3 astrocytes. To assess the impacts of tau propagation effects, we next developed a sophisticated and sensitive assay utilizing cortical neurons derived from human iPSCs, previously generated from donors of both sexes. We showed that APOE3Ch/3Ch astrocytes effectively mitigated tau propagation within iPSC-derived neurons. This study provides important experimental evidence of the characteristic functions exhibited by APOE3Ch/3Ch astrocytes, thereby offering valuable insights for the advancement of novel clinical interventions in AD research.

Astrocytic APOE4 genotype-mediated negative impacts on synaptic architecture in human pluripotent stem cell model.

The APOE4 genotype is the strongest risk factor for the pathogenesis of sporadic Alzheimer's disease (AD), but the detailed molecular mechanism of APOE4-mediated synaptic impairment remains to be determined. In this study, we generated a human astrocyte model carrying the APOE3 or APOE4 genotype using human induced pluripotent stem cells (iPSCs) in which isogenic APOE4 iPSCs were genome edited from healthy control APOE3 iPSCs. Next, we demonstrated that the astrocytic APOE4 genotype negatively affects dendritic spine dynamics in a co-culture system with primary neurons. Transcriptome analysis revealed an increase of EDIL3, an extracellular matrix glycoprotein, in human APOE4 astrocytes, which could underlie dendritic spine reduction in neuronal cultures. Accordingly, postmortem AD brains carrying the APOE4 allele have elevated levels of EDIL3 protein deposits within amyloid plaques. Together, these results demonstrate the novel deleterious effect of human APOE4 astrocytes on synaptic architecture and may help to elucidate the mechanism of APOE4-linked AD pathogenesis.

Casein kinase 1δ/ε phosphorylates fused in sarcoma (FUS) and ameliorates FUS-mediated neurodegeneration.

Aberrant cytoplasmic accumulation of an RNA-binding protein, fused in sarcoma (FUS), characterizes the neuropathology of subtypes of ALS and frontotemporal lobar degeneration, although the effects of post-translational modifications of FUS, especially phosphorylation, on its neurotoxicity have not been fully characterized. Here, we show that casein kinase 1δ (CK1δ) phosphorylates FUS at 10 serine/threonine residues in vitro using mass spectrometric analyses. We also show that phosphorylation by CK1δ or CK1ε significantly increased the solubility of FUS in human embryonic kidney 293 cells. In transgenic Drosophila that overexpress wt or P525L ALS-mutant human FUS in the retina or in neurons, we found coexpression of human CK1δ or its Drosophila isologue Dco in the photoreceptor neurons significantly ameliorated the observed retinal degeneration, and neuronal coexpression of human CK1δ extended fly life span. Taken together, our data suggest a novel regulatory mechanism of the assembly and toxicity of FUS through CK1δ/CK1ε-mediated phosphorylation, which could represent a potential therapeutic target in FUS proteinopathies.

Lipid flippase dysfunction as a therapeutic target for endosomal anomalies in Alzheimer's disease.

Endosomal anomalies because of vesicular traffic impairment have been indicated as an early pathology of Alzheimer'| disease (AD). However, the mechanisms and therapeutic targets remain unclear. We previously reported that ßCTF, one of the pathogenic metabolites of APP, interacts with TMEM30A. TMEM30A constitutes a lipid flippase with P4-ATPase and regulates vesicular trafficking through the asymmetric distribution of phospholipids. Therefore, the alteration of lipid flippase activity in AD pathology has got attention. Herein, we showed that the interaction between ßCTF and TMEM30A suppresses the physiological formation and activity of lipid flippase in AD model cells, A7, and AppNL-G-F/NL-G-F model mice. Furthermore, the T-RAP peptide derived from the ßCTF binding site of TMEM30A improved endosomal anomalies, which could be a result of the restored lipid flippase activity. Our results provide insights into the mechanisms of vesicular traffic impairment and suggest a therapeutic target for AD.

Glymphatic system clears extracellular tau and protects from tau aggregation and neurodegeneration.

Accumulation of tau has been implicated in various neurodegenerative diseases termed tauopathies. Tau is a microtubule-associated protein but is also actively released into the extracellular fluids including brain interstitial fluid and cerebrospinal fluid (CSF). However, it remains elusive whether clearance of extracellular tau impacts tau-associated neurodegeneration. Here, we show that aquaporin-4 (AQP4), a major driver of the glymphatic clearance system, facilitates the elimination of extracellular tau from the brain to CSF and subsequently to deep cervical lymph nodes. Strikingly, deletion of AQP4 not only elevated tau in CSF but also markedly exacerbated phosphorylated tau deposition and the associated neurodegeneration in the brains of transgenic mice expressing P301S mutant tau. The current study identified the clearance pathway of extracellular tau in the central nervous system, suggesting that glymphatic clearance of extracellular tau is a novel regulatory mechanism whose impairment contributes to tau aggregation and neurodegeneration.

ALS-linked cytoplasmic FUS assemblies are compositionally different from physiological stress granules and sequester hnRNPA3, a novel modifier of FUS toxicity.

Formation of cytoplasmic RNA-protein structures called stress granules (SGs) is a highly conserved cellular response to stress. Abnormal metabolism of SGs may contribute to the pathogenesis of (neuro)degenerative diseases such as amyotrophic lateral sclerosis (ALS). Many SG proteins are affected by mutations causative of these conditions, including fused in sarcoma (FUS). Mutant FUS variants have high affinity to SGs and also spontaneously form de novo cytoplasmic RNA granules. Mutant FUS-containing assemblies (mFAs), often called "pathological SGs", are proposed to play a role in ALS-FUS pathogenesis. However, structural differences between mFAs and physiological SGs remain largely unknown therefore it is unclear whether mFAs can functionally substitute for SGs and how they affect cellular stress responses. Here we used affinity purification to isolate mFAs and physiological SGs and compare their protein composition. We found that proteins within mFAs form significantly more physical interactions than those in SGs however mFAs fail to recruit many factors involved in signal transduction. Furthermore, we found that proteasome subunits and certain nucleocytoplasmic transport factors are depleted from mFAs, whereas translation elongation, mRNA surveillance and splicing factors as well as mitochondrial proteins are enriched in mFAs, as compared to SGs. Validation experiments for a mFA-specific protein, hnRNPA3, confirmed its RNA-dependent interaction with FUS and its sequestration into FUS inclusions in cultured cells and in a FUS transgenic mouse model. Silencing of the Drosophila hnRNPA3 ortholog was deleterious and potentiated human FUS toxicity in the retina of transgenic flies. In conclusion, we show that SG-like structures formed by mutant FUS are structurally distinct from SGs, prone to persistence, likely cannot functionally replace SGs, and affect a spectrum of cellular pathways in stressed cells. Results of our study support a pathogenic role for cytoplasmic FUS assemblies in ALS-FUS.

Long non-coding RNA NEAT1_1 ameliorates TDP-43 toxicity in in vivo models of TDP-43 proteinopathy.

Pathological changes involving TDP-43 protein ('TDP-43 proteinopathy') are typical for several neurodegenerative diseases, including frontotemporal lobar degeneration (FTLD). FTLD-TDP cases are characterized by increased binding of TDP-43 to an abundant lncRNA, NEAT1, in the cortex. However it is unclear whether enhanced TDP-43-NEAT1 interaction represents a protective mechanism. We show that accumulation of human TDP-43 leads to upregulation of the constitutive NEAT1 isoform, NEAT1_1, in cultured cells and in the brains of transgenic mice. Further, we demonstrate that overexpression of NEAT1_1 ameliorates TDP-43 toxicity in Drosophila and yeast models of TDP-43 proteinopathy. Thus, NEAT1_1 upregulation may be protective in TDP-43 proteinopathies affecting the brain. Approaches to boost NEAT1_1 expression in the CNS may prove useful in the treatment of these conditions.

Collagenous Alzheimer amyloid plaque component impacts on the compaction of amyloid-ß plaques.

Massive deposition of amyloid ß peptides (Aß) as senile plaques (SP) characterizes the brain pathology of Alzheimer's disease (AD). SPs exhibit a variety of morphologies, although little is known about the SP components that determine their morphology. Collagenous Alzheimer amyloid plaque component (CLAC) is one of the major non-Aß proteinaceous components of SP amyloid in AD brains. Here we show that overexpression of CLAC precursor (CLAC-P) in the brains of APP transgenic mice results in a significant remodeling of amyloid pathology, i.e., reduction in diffuse-type amyloid plaques and an increase in compact plaques laden with thioflavin S-positive amyloid cores. In vivo microdialysis revealed a significant decrease in Aß in the brain interstitial fluid of CLAC-P/APP double transgenic mice compared with APP transgenic mice. These findings implicate CLAC in the compaction of Aß in amyloid plaques and the brain dynamics of Aß.

Behavioral and electrophysiological evidence for a neuroprotective role of aquaporin-4 in the 5xFAD transgenic mice model.

Aquaporin-4 (AQP4) has been suggested to be involved in the pathogenesis of neurodegenerative diseases including Alzheimer's disease (AD), which may be due to the modulation of neuroinflammation or the impairment of interstitial fluid bulk flow system in the central nervous system. Here, we show an age-dependent impairment of several behavioral outcomes in 5xFAD AQP4 null mice. Twenty-four-hour video recordings and computational analyses of their movement revealed that the nighttime motion of AQP4-deficient 5xFAD mice was progressively reduced between 20 and 36 weeks of age, with a sharp deterioration occurring between 30 and 32 weeks. This reduction in nighttime motion was accompanied by motor dysfunction and epileptiform neuronal activities, demonstrated by increased abnormal spikes by electroencephalography. In addition, all AQP4-deficient 5xFAD mice exhibited convulsions at least once during the period of the analysis. Interestingly, despite such obvious phenotypes, parenchymal amyloid ß (Aß) deposition, reactive astrocytosis, and activated microgliosis surrounding amyloid plaques were unchanged in the AQP4-deficient 5xFAD mice relative to 5xFAD mice. Taken together, our data indicate that AQP4 deficiency greatly accelerates an age-dependent deterioration of neuronal function in 5xFAD mice associated with epileptiform neuronal activity without significantly altering Aß deposition or neuroinflammation in this mouse model. We therefore propose that there exists another pathophysiological phase in AD which follows amyloid plaque deposition and neuroinflammation and is sensitive to AQP4 deficiency.

Roles of Collagen XXV and Its Putative Receptors PTPσ/δ in Intramuscular Motor Innervation and Congenital Cranial Dysinnervation Disorder.

Intramuscular motor innervation is an essential process in neuromuscular development. Recently, mutations in COL25A1, encoding CLAC-P/collagen XXV, have been linked to the development of a congenital cranial dysinnervation disorder (CCDD). Yet the molecular mechanisms of intramuscular innervation and the etiology of CCDD related to COL25A1 have remained elusive. Here, we report that muscle-derived collagen XXV is indispensable for intramuscular innervation. In developing skeletal muscles, Col25a1 expression is tightly regulated by muscle excitation. In vitro and cell-based assays reveal a direct interaction between collagen XXV and receptor protein tyrosine phosphatases (PTPs) σ and δ. Motor explant assays show that expression of collagen XXV in target cells attracts motor axons, but this is inhibited by exogenous PTPσ/δ. CCDD mutations attenuate motor axon attraction by reducing collagen XXV-PTPσ/δ interaction. Overall, our study identifies PTPσ/δ as putative receptors for collagen XXV, implicating collagen XXV and PTPσ/δ in intramuscular innervation and a developmental ocular motor disorder.

Calcium-responsive transactivator (CREST) toxicity is rescued by loss of PBP1/ATXN2 function in a novel yeast proteinopathy model and in transgenic flies.

Proteins associated with familial neurodegenerative disease often aggregate in patients' neurons. Several such proteins, e.g. TDP-43, aggregate and are toxic when expressed in yeast. Deletion of the ATXN2 ortholog, PBP1, reduces yeast TDP-43 toxicity, which led to identification of ATXN2 as an amyotrophic lateral sclerosis (ALS) risk factor and therapeutic target. Likewise, new yeast neurodegenerative disease models could facilitate identification of other risk factors and targets. Mutations in SS18L1, encoding the calcium-responsive transactivator (CREST) chromatin-remodeling protein, are associated with ALS. We show that CREST is toxic in yeast and forms nuclear and occasionally cytoplasmic foci that stain with Thioflavin-T, a dye indicative of amyloid-like protein. Like the yeast chromatin-remodeling factor SWI1, CREST inhibits silencing of FLO genes. Toxicity of CREST is enhanced by the [PIN+] prion and reduced by deletion of the HSP104 chaperone required for the propagation of many yeast prions. Likewise, deletion of PBP1 reduced CREST toxicity and aggregation. In accord with the yeast data, we show that the Drosophila ortholog of human ATXN2, dAtx2, is a potent enhancer of CREST toxicity. Downregulation of dAtx2 in flies overexpressing CREST in retinal ganglion cells was sufficient to largely rescue the severe degenerative phenotype induced by human CREST. Overexpression caused considerable co-localization of CREST and PBP1/ATXN2 in cytoplasmic foci in both yeast and mammalian cells. Thus, co-aggregation of CREST and PBP1/ATXN2 may serve as one of the mechanisms of PBP1/ATXN2-mediated toxicity. These results extend the spectrum of ALS associated proteins whose toxicity is regulated by PBP1/ATXN2, suggesting that therapies targeting ATXN2 may be effective for a wide range of neurodegenerative diseases.

Characterization of the unique In Vitro effects of unsaturated fatty acids on the formation of amyloid ß fibrils.

Accumulation of amyloid ß (Aß) peptides, the major component of amyloid fibrils in senile plaques, is one of the main causes of Alzheimer's disease. Docosahexaenoic acid (DHA) is a fatty acid abundant in the brain, and is reported to have protective effects against Alzheimer's disease, although the mechanistic effects of DHA against Alzheimer's pathophysiology remain unclear. Because dietary supplementation of DHA in Aß precursor protein transgenic mice ameliorates Aß pathology and behavioral deficits, we hypothesize that DHA may affect the fibrillization and deposition of Aß. Here we studied the effect of different types of fatty acids on Aß fibril formation by in vitro Aß fibrillization assay. Formation of amyloid fibrils consists of two steps, i.e., the initial nucleation phase and the following elongation phase. We found that unsaturated fatty acids, especially DHA, accelerated the formation of Aß fibrils with a unique short and curved morphology in its nucleation phase, which did not elongate further into the long and straight, mature Aß fibrils. Addition of DHA afterwards did not modify the morphology of the mature Aß(1-40) fibrils. The short and curved Aß fibrils formed in the presence of DHA did not facilitate the elongation phase of Aß fibril formation, suggesting that DHA promotes the formation of "off-pathway" conformers of Aß. Our study unravels a possible mechanism of how DHA acts protectively against the pathophysiology of Alzheimer's disease.

Differential effects of diet- and genetically-induced brain insulin resistance on amyloid pathology in a mouse model of Alzheimer's disease.

BACKGROUND: Based on epidemiological and experimental studies, type 2 diabetes mellitus (T2DM), especially insulin resistance that comprises the core mechanism of T2DM, has been recognized as a significant risk factor for Alzheimer's disease (AD). Studies in humans and diabetic AD model mice have indicated a correlation between insulin resistance and increased amyloid deposition in the brain. Paradoxically, mice with targeted disruption of genes involved in the insulin signaling pathway showed protective effects against the AD-related pathology. These conflicting observations raise an issue as to the relationship between dysregulation of insulin signaling and AD pathophysiology. METHODS: To study the causal relations and molecular mechanisms underlying insulin resistance-induced exacerbation of amyloid pathology, we investigated the chronological changes in the development of insulin resistance and amyloid pathology in two independent insulin-resistant AD mouse models, i.e., long-term high-fat diet (HFD) feeding and genetic disruption of Irs2, in combination with dietary interventions. In addition to biochemical and histopathological analyses, we examined the in vivo dynamics of brain amyloid-ß (Aß) and insulin by microdialysis technique. RESULTS: HFD-fed diabetic AD model mice displayed a reduced brain response to peripheral insulin stimulation and a decreased brain to plasma ratio of insulin during the hyperinsulinemic clamp. Diet-induced defective insulin action in the brain was accompanied by a decreased clearance of the extracellular Aß in vivo and an exacerbation of brain amyloid pathology. These noxious effects of the HFD both on insulin sensitivity and on Aß deposition in brains were reversibly attenuated by dietary interventions. Importantly, HFD feeding accelerated Aß deposition also in the brains of IRS-2-deficient AD mice. CONCLUSIONS: Our results suggested a causal and reversible association of brain Aß metabolism and amyloid pathology by diet-dependent, but not genetically-induced, insulin-resistance. These observations raise the possibility that the causal factors of insulin resistance, e.g., metabolic stress or inflammation induced by HFD feeding, but not impaired insulin signaling per se, might be directly involved in the acceleration of amyloid pathology in the brain.

Chronic cerebral hypoperfusion shifts the equilibrium of amyloid ß oligomers to aggregation-prone species with higher molecular weight.

Epidemiological studies have shown that atherosclerotic risk factors accelerate the pathological process underlying Alzheimer's disease (AD) via chronic cerebral hypoperfusion. In this study, we aimed to clarify the mechanisms by which cerebral hypoperfusion may exacerbate AD pathology. We applied bilateral common carotid artery stenosis (BCAS) to a mice model of AD and evaluated how the equilibrium of amyloid ß oligomers respond to hypoperfusion. BCAS accelerated amyloid ß (Aß) convergence to the aggregation seed, facilitating the growth of Aß plaques, but without changing the total Aß amount in the brain. Furthermore, Aß oligomers with high molecular weight increased in the brain of BCAS-operated mice. Considering Aß is in an equilibrium among monomeric, oligomeric, and aggregation forms, our data suggest that cerebral hypoperfusion after BCAS shifted this equilibrium to a state where a greater number of Aß molecules participate in Aß assemblies to form aggregation-prone Aß oligomers with high molecular weight. The reduced blood flow in the cerebral arteries due to BCAS attenuated the dynamics of the interstitial fluid leading to congestion, which may have facilitated Aß aggregation. We suggest that cerebral hypoperfusion may accelerate AD by enhancing the tendency of Aß to become aggregation-prone.

Patterns and severity of vascular amyloid in Alzheimer's disease associated with duplications and missense mutations in APP gene, Down syndrome and sporadic Alzheimer's disease.

In this study, we have compared the severity of amyloid plaque formation and cerebral amyloid angiopathy (CAA), and the subtype pattern of CAA pathology itself, between APP genetic causes of AD (APPdup, APP mutations), older individuals with Down syndrome (DS) showing the pathology of Alzheimer's disease (AD) and individuals with sporadic (early and late onset) AD (sEOAD and sLOAD, respectively). The aim of this was to elucidate important group differences and to provide mechanistic insights related to clinical and neuropathological phenotypes. Since lipid and cholesterol metabolism is implicated in AD as well as vascular disease, we additionally aimed to explore the role of APOE genotype in CAA severity and subtypes. Plaque formation was greater in DS and missense APP mutations than in APPdup, sEOAD and sLOAD cases. Conversely, CAA was more severe in APPdup and missense APP mutations, and in DS, compared to sEOAD and sLOAD. When stratified by CAA subtype from 1 to 4, there were no differences in plaque scores between the groups, though in patients with APPdup, APP mutations and sEOAD, types 2 and 3 CAA were more common than type 1. Conversely, in DS, sLOAD and controls, type 1 CAA was more common than types 2 and 3. APOE ε4 allele frequency was greater in sEOAD and sLOAD compared to APPdup, missense APP mutations, DS and controls, and varied between each of the CAA phenotypes with APOE ε4 homozygosity being more commonly associated with type 3 CAA than types 1 and 2 CAA in sLOAD and sEOAD. The differing patterns in CAA within individuals of each group could be a reflection of variations in the efficiency of perivascular drainage, this being less effective in types 2 and 3 CAA leading to a greater burden of CAA in parenchymal arteries and capillaries. Alternatively, as suggested by immunostaining using carboxy-terminal specific antibodies, it may relate to the relative tissue burdens of the two major forms of Aß, with higher levels of Aß40 promoting a more 'aggressive' form of CAA, and higher levels of Aß42(3) favouring a greater plaque burden. Possession of APOE ε4 allele, especially ε4 homozygosity, favours development of CAA generally, and as type 3 particularly, in sEOAD and sLOAD.

Self-assembly of FUS through its low-complexity domain contributes to neurodegeneration.

Aggregation of fused in sarcoma (FUS) protein, and mutations in FUS gene, are causative to a range of neurodegenerative disorders including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. To gain insights into the molecular mechanism whereby FUS causes neurodegeneration, we generated transgenic Drosophila melanogaster overexpressing human FUS in the photoreceptor neurons, which exhibited mild retinal degeneration. Expression of familial ALS-mutant FUS aggravated the degeneration, which was associated with an increase in cytoplasmic localization of FUS. A carboxy-terminally truncated R495X mutant FUS also was localized in cytoplasm, whereas the degenerative phenotype was diminished. Double expression of R495X and wild-type FUS dramatically exacerbated degeneration, sequestrating wild-type FUS into cytoplasmic aggregates. Notably, replacement of all tyrosine residues within the low-complexity domain, which abolished self-assembly of FUS, completely eliminated the degenerative phenotypes. Taken together, we propose that self-assembly of FUS through its low-complexity domain contributes to FUS-induced neurodegeneration.

Neuron-specific methylome analysis reveals epigenetic regulation and tau-related dysfunction of BRCA1 in Alzheimer's disease.

Alzheimer's disease (AD) is a chronic neurodegenerative disease characterized by pathology of accumulated amyloid ß (Aß) and phosphorylated tau proteins in the brain. Postmortem degradation and cellular complexity within the brain have limited approaches to molecularly define the causal relationship between pathological features and neuronal dysfunction in AD. To overcome these limitations, we analyzed the neuron-specific DNA methylome of postmortem brain samples from AD patients, which allowed differentially hypomethylated region of the BRCA1 promoter to be identified. Expression of BRCA1 was significantly up-regulated in AD brains, consistent with its hypomethylation. BRCA1 protein levels were also elevated in response to DNA damage induced by Aß. BRCA1 became mislocalized to the cytoplasm and highly insoluble in a tau-dependent manner, resulting in DNA fragmentation in both in vitro cellular and in vivo mouse models. BRCA1 dysfunction under Aß burden is consistent with concomitant deterioration of genomic integrity and synaptic plasticity. The Brca1 promoter region of AD model mice brain was similarly hypomethylated, indicating an epigenetic mechanism underlying BRCA1 regulation in AD. Our results suggest deterioration of DNA integrity as a central contributing factor in AD pathogenesis. Moreover, these data demonstrate the technical feasibility of using neuron-specific DNA methylome analysis to facilitate discovery of etiological candidates in sporadic neurodegenerative diseases.

Soluble oligomeric amyloid-ß induces calcium dyshomeostasis that precedes synapse loss in the living mouse brain.

BACKGROUND: Amyloid-ß oligomers (oAß) are thought to mediate neurotoxicity in Alzheimer's disease (AD), and previous studies in AD transgenic mice suggest that calcium dysregulation may contribute to these pathological effects. Even though AD mouse models remain a valuable resource to investigate amyloid neurotoxicity, the concomitant presence of soluble Aß species, fibrillar Aß, and fragments of amyloid precursor protein (APP) complicate the interpretation of the phenotypes. METHOD: To explore the specific contribution of soluble oligomeric Aß (oAß) to calcium dyshomeostasis and synaptic morphological changes, we acutely exposed the healthy mouse brain, at 3 to 6 months of age, to naturally occurring soluble oligomers and investigated their effect on calcium levels using in vivo multiphoton imaging. RESULTS: We observed a dramatic increase in the levels of neuronal resting calcium, which was dependent upon extracellular calcium influx and activation of NMDA receptors. Ryanodine receptors, previously implicated in AD models, did not appear to be primarily involved using this experimental setting. We used the high resolution cortical volumes acquired in-vivo to measure the effect on synaptic densities and observed that, while spine density remained stable within the first hour of oAß exposure, a significant decrease in the number of dendritic spines was observed 24 h post treatment, despite restoration of intraneuronal calcium levels at this time point. CONCLUSIONS: These observations demonstrate a specific effect of oAß on NMDA-mediated calcium influx, which triggers synaptic collapse in vivo. Moreover, this work leverages a method to quantitatively measure calcium concentration at the level of neuronal processes, cell bodies and single synaptic elements repeatedly and thus can be applicable to testing putative drugs and/or other intervention methodologies.

Familial Amyotrophic Lateral Sclerosis-linked Mutations in Profilin 1 Exacerbate TDP-43-induced Degeneration in the Retina of Drosophila melanogaster through an Increase in the Cytoplasmic Localization of TDP-43.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive and selective loss of motor neurons. Causative genes for familial ALS (fALS), e.g. TARDBP or FUS/TLS, have been found, among which mutations within the profilin 1 (PFN1) gene have recently been identified in ALS18. To elucidate the mechanism whereby PFN1 mutations lead to neuronal death, we generated transgenic Drosophila melanogaster overexpressing human PFN1 in the retinal photoreceptor neurons. Overexpression of wild-type or fALS mutant PFN1 caused no degenerative phenotypes in the retina. Double overexpression of fALS mutant PFN1 and human TDP-43 markedly exacerbated the TDP-43-induced retinal degeneration, i.e. vacuolation and thinning of the retina, whereas co-expression of wild-type PFN1 did not aggravate the degenerative phenotype. Notably, co-expression of TDP-43 with fALS mutant PFN1 increased the cytoplasmic localization of TDP-43, the latter remaining in nuclei upon co-expression with wild-type PFN1, whereas co-expression of TDP-43 lacking the nuclear localization signal with the fALS mutant PFN1 did not aggravate the retinal degeneration. Knockdown of endogenous Drosophila PFN1 did not alter the degenerative phenotypes of the retina in flies overexpressing wild-type TDP-43 These data suggest that ALS-linked PFN1 mutations exacerbate TDP-43-induced neurodegeneration in a gain-of-function manner, possibly by shifting the localization of TDP-43 from nuclei to cytoplasm.