Genetic Reduction of Insulin Signaling Mitigates Amyloid-ß Deposition by Promoting Expression of Extracellular Matrix Proteins in the Brain.

The insulin/IGF-1 signaling (IIS) regulates a wide range of biological processes, including aging and lifespan, and has also been implicated in the pathogenesis of Alzheimer's disease (AD). We and others have reported that reduced signaling by genetic ablation of the molecules involved in IIS (e.g., insulin receptor substrate 2 [IRS-2]) markedly mitigates amyloid plaque formation in the brains of mouse models of AD, although the molecular underpinnings of the amelioration remain unsolved. Here, we revealed, by a transcriptomic analysis of the male murine cerebral cortices, that the expression of genes encoding extracellular matrix (ECM) was significantly upregulated by the loss of IRS-2. Insulin signaling activity negatively regulated the phosphorylation of Smad2 and Smad3 in the brain, and suppressed TGF-ß/Smad-dependent expression of a subset of ECM genes in brain-derived cells. The ECM proteins inhibited Aß fibril formation in vitro, and IRS-2 deficiency suppressed the aggregation process of Aß in the brains of male APP transgenic mice as revealed by injection of aggregation seeds in vivo Our results propose a novel mechanism in AD pathophysiology whereby IIS modifies Aß aggregation and amyloid pathology by altering the expression of ECM genes in the brain.SIGNIFICANCE STATEMENT The insulin/IGF-1 signaling (IIS) has been recognized as a regulator of aging, a leading risk factor for the onset of Alzheimer's disease (AD). In AD mouse models, genetic deletion of key IIS molecules markedly reduces the amyloid plaque formation in the brain, although the molecular underpinnings of this amelioration remain elusive. We found that the deficiency of insulin receptor substrate 2 leads to an increase in the expression of various extracellular matrices (ECMs) in the brain, potentially through TGF-ß/Smad signaling. Furthermore, some of those ECMs exhibited the potential to inhibit amyloid plaque accumulation by disrupting the formation of Aß fibrils. This study presents a novel mechanism by which IIS regulates Aß accumulation, which may involve altered brain ECM expression.

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.

Differential involvement of insulin receptor substrate (IRS)-1 and IRS-2 in brain insulin signaling is associated with the effects on amyloid pathology in a mouse model of Alzheimer's disease.

Insulin signaling has been implicated in the metabolism as well as aging and longevity. Type 2 diabetes mellitus and its core pathology, insulin resistance, has also been implicated in the development of Alzheimer's disease (AD) and amyloid-ß deposition in humans. By contrast, genetic ablation of the insulin/IGF-1 signaling (IIS) pathway components, e.g. insulin receptor substrate (IRS)-2, has been documented to suppress amyloid-ß accumulation in the brains of transgenic mice overexpressing AD mutant ß-amyloid precursor protein (APP). Therefore, the brain IIS may be a key modifiable molecular target in the pathophysiology of AD. IRS-1 and IRS-2 are critical nodes in IIS as substrates for insulin receptor and IGF-1 receptor, although the functional differences between IRS-1 and IRS-2 in the adult brain are yet to be explored. To examine their relative contribution to the brain IIS activity and AD pathomechanism, we generated APP transgenic mice lacking either IRS-1 or IRS-2. IRS-1 deficiency had little effects on the brain IIS pathway associated with compensatory activation of IRS-2, whereas IRS-2 deficiency was not fully compensated by activation of IRS-1, and the downstream activation of Akt also was significantly compromised. Pathological analyses of the cortical tissues showed that the biochemical levels of soluble and insoluble amyloid-ß, the amyloid-ß histopathology, and tau phosphorylation were not affected by the absence of IRS-1, in contrast to the marked alteration in IRS-2 deleted mice. These results suggest the predominance of IRS-2 in the brain IIS, and support the hypothesis that reduced IIS exerts anti-amyloid effects in the brain.

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.

Transmembrane Collagens in Neuromuscular Development and Disorders.

Neuromuscular development is a multistep process and involves interactions among various extracellular and transmembrane molecules that facilitate the precise targeting of motor axons to synaptogenic regions of the target muscle. Collagenous proteins with transmembrane domains have recently emerged as molecules that play essential roles in multiple aspects of neuromuscular formation. Membrane-associated collagens with interrupted triple helices (MACITs) are classified as an unconventional subtype of the collagen superfamily and have been implicated in cell adhesion in a variety of tissues, including the neuromuscular system. Collagen XXV, the latest member of the MACITs, plays an essential role in motor axon growth within the developing muscle. In humans, loss-of-function mutations of collagen XXV result in developmental ocular motor disorders. In contrast, collagen XIII contributes to the formation and maintenance of neuromuscular junctions (NMJs), and disruption of its function leads to the congenital myasthenic syndrome. Transmembrane collagens are conserved not only in mammals but also in organisms such as C. elegans, where a single MACIT, COL-99, has been documented to function in motor innervation. Furthermore, in C. elegans, a collagen-like transmembrane protein, UNC-122, is implicated in the structural and functional integrity of the NMJ. This review article summarizes recent advances in understanding the roles of transmembrane collagens and underlying molecular mechanisms in multiple aspects of neuromuscular development and disorders.

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.

Author Correction: Collagen XXV promotes myoblast fusion during myogenic differentiation and muscle formation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

Collagen XXV promotes myoblast fusion during myogenic differentiation and muscle formation.

Fusion of myoblasts into multinucleated myofibers is crucial for skeletal muscle development and regeneration. However, the mechanisms controlling this process remain to be determined. Here we identified the involvement of a new extracellular matrix protein in myoblast fusion. Collagen XXV is a transmembrane-type collagen highly transcribed during early myogenesis when primary myofibers form. Limb muscles of E12.5 and E14.5 Col25a1-/- embryos show a clear defect in the formation of multinucleated myofibers. In cell culture, the cleaved soluble extracellular domain of the collagen XXV is sufficient to promote the formation of highly multinucleated myofibers. Col25a1 is transiently expressed during myogenic differentiation and Col25a1 transcripts are down-regulated in multinucleated myofibers by a muscle-specific microRNA, miR-499. Altogether, these findings indicate that collagen XXV is required in vivo and in vitro for the fusion of myoblasts into myofibers and give further evidence that microRNAs participate to the regulation of this process.

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.

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.

Chronic optogenetic activation augments aß pathology in a mouse model of Alzheimer disease.

In vivo experimental evidence indicates that acute neuronal activation increases Aß release from presynaptic terminals, whereas long-term effects of chronic synaptic activation on Aß pathology remain unclear. To address this issue, we adopted optogenetics and transduced stabilized step-function opsin, a channelrhodopsin engineered to elicit a long-lasting neuronal hyperexcitability, into the hippocampal perforant pathway of APP transgenic mice. In vivo microdialysis revealed a ∼24% increase in the hippocampal interstitial fluid Aß42 levels immediately after acute light activation. Five months of chronic optogenetic stimulation increased Aß burden specifically in the projection area of the perforant pathway (i.e., outer molecular layer of the dentate gyrus) of the stimulated side by ∼2.5-fold compared with that in the contralateral side. Epileptic seizures were observed during the course of chronic stimulation, which might have partly contributed to the Aß pathology. These findings implicate functional abnormalities of specific neuronal circuitry in Aß pathology and Alzheimer disease.

CLAC-P/collagen type XXV is required for the intramuscular innervation of motoneurons during neuromuscular development.

Formation of proper neuromuscular connections is a process coordinated by both motoneuron-intrinsic and target-dependent programs. Under these programs, motoneurons innervate target muscles, escape programmed cell death during fetal development, and form neuromuscular junctions (NMJ). Although a number of studies have revealed molecules involved in axon guidance to target muscles and NMJ formation, little is known about the molecular mechanisms linking intramuscular innervation and target-derived trophic factor-dependent prevention of motoneuron apoptosis. Here we studied the physiological function of CLAC-P/collagen XXV, a transmembrane-type collagen originally identified as a component of senile plaque amyloid of Alzheimer's disease brains, by means of generating Col25a1-deficient (KO) mice. Col25a1 KO mice died immediately after birth of respiratory failure. In Col25a1 KO mice, motor axons projected properly toward the target muscles but failed to elongate and branch within the muscle, followed by degeneration of axons. Failure of muscular innervation in Col25a1 KO mice led to excessive apoptosis during development, resulting in almost complete and exclusive loss of spinal motoneurons and immaturity in skeletal muscle development. Bax deletion in Col25a1 KO mice rescued motoneurons from apoptosis, although motor axons remained halted around the muscle entry site. Furthermore, these motoneurons were positive for phosphorylated c-Jun, an indicator of insufficient supply of target-derived survival signals. Together, these observations indicate that CLAC-P/collagen XXV is a novel essential factor that regulates the initial phase of intramuscular motor innervation, which is required for subsequent target-dependent motoneuron survival and NMJ formation during development.

RNA binding mediates neurotoxicity in the transgenic Drosophila model of TDP-43 proteinopathy.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive and selective loss of motor neurons. The discovery of mutations in the gene encoding an RNA-binding protein, TAR DNA-binding protein of 43 kD (TDP-43), in familial ALS, strongly implicated abnormalities in RNA processing in the pathogenesis of ALS, although the mechanisms whereby TDP-43 leads to neurodegeneration remain elusive. To clarify the mechanism of degeneration caused by TDP-43, we generated transgenic Drosophila melanogaster expressing a series of systematically modified human TDP-43 genes in the retinal photoreceptor neurons. Overexpression of wild-type TDP-43 resulted in vacuolar degeneration of the photoreceptor neurons associated with thinning of the retina, which was significantly exacerbated by mutations of TDP-43 linked to familial ALS or disrupting its nuclear localization signal (NLS). Remarkably, these degenerative phenotypes were completely normalized by addition of a mutation or deletion of the RNA recognition motif that abolishes the RNA binding ability of TDP-43. Altogether, our results suggest that RNA binding is key to the neurodegeneration caused by overexpression of TDP-43, and that abnormalities in RNA processing may be crucial to the pathogenesis of TDP-43 proteinopathy.

Analysis of the gamma-secretase interactome and validation of its association with tetraspanin-enriched microdomains.

Gamma-secretase, an aspartyl protease that belongs to the iCLiPs (intramembrane cleaving proteases) family, is a multiprotein complex that consists of presenilin (PS), nicastrin (NCT), Aph-1 and Pen-2 (ref. 1). It is responsible for generation of the beta-amyloid peptide (Abeta), the primary component of senile plaques in the brains of patients with Alzheimer's disease. Although the four components are necessary and sufficient for gamma-secretase activity, additional proteins are possibly involved in its regulation. Consequently, we purified proteins associated with the active gamma-secretase complex from reconstituted PS-deficient fibroblasts, using tandem affinity purification (TAP) and identified a series of proteins that transiently interact with the gamma-secretase complex and are probably involved in complex maturation, membrane trafficking and, importantly, the tetraspanin web. Tetraspanins form detergent-resistant microdomains in the cell membrane and regulate cell adhesion, cell signalling and proteolysis. Association of the gamma-secretase complex with tetraspanin-enriched microdomains provides an explanation for the previously documented localization of gamma-secretase to raft-like domains. Thus, these studies suggest that maintenance of the integrity of tetraspanin microdomains contributes to the refinement of proteolytic activity of the gamma-secretase complex.

Presenilins: members of the gamma-secretase quartets, but part-time soloists too.

The presenilins in combination with other proteins generate different gamma-secretases, which are involved in the regulated intramembrane proteolysis of a variety of proteins. Understanding the specificity and regulation of these proteases will potentially lead to novel therapeutics for Alzheimer's disease and cancer. Presenilins appear also to exert additional functions outside of the gamma-secretase quartets, which needs further investigation.

CLAC binds to amyloid beta peptides through the positively charged amino acid cluster within the collagenous domain 1 and inhibits formation of amyloid fibrils.

CLAC (collagenous Alzheimer amyloid plaque component) is a proteolytic fragment derived from a novel membrane-bound collagen, CLAC-P/collagen type XXV, that deposits in senile plaques associated with amyloid beta peptides (Abeta) in the brains of patients with Alzheimer's disease. We previously showed that CLAC binds to the fibrillized form of Abeta in vitro, although the mechanism and the subdomains that mediate interaction of CLAC with Abeta as well as the effect of binding of CLAC on amyloid fibril formation remain unknown. Here we show that the collagenous domain 1 of CLAC, which is rich in positively charged amino acid residues, mediates its interaction with Abeta and that this binding is mediated by an electrostatic interaction and requires formation of the triple helix structure of CLAC. The soluble form of CLAC purified from the media of cells transfected with CLAC-P inhibited fibrillization of Abeta in vitro, especially in its elongation phase. These results suggest the anti-amyloidogenic roles of CLAC in the pathophysiology of Alzheimer's disease.

Mostly separate distributions of CLAC- versus Abeta40- or thioflavin S-reactivities in senile plaques reveal two distinct subpopulations of beta-amyloid deposits.

Collagenous Alzheimer amyloid plaque component (CLAC) is a unique non-Abeta amyloid component of senile plaques (SP) derived from a transmembrane collagen termed CLAC-precursor. Here we characterize the chronological and spatial relationship of CLAC with other features of SP amyloid in the brains of patients with Alzheimer's disease (AD), Down syndrome (DS), and of PSAPP transgenic mice. In AD and DS cerebral cortex, CLAC invariably colocalized with Abeta42 but often lacked Abeta40- or thioflavin S (thioS)-reactivities. Immunoelectron microscopy of CLAC-positive SP showed labeling of fibrils that are more loosely dispersed compared to typical amyloid fibrils in CLAC-negative SP. In DS cerebral cortex, diffuse plaques in young patients were negative for CLAC, whereas a subset of SP became CLAC-positive in patients aged 35 to 50 years, before the appearance of Abeta40. In DS cases over 50 years of age, Abeta40-positive SP dramatically increased, whereas CLAC burden remained at a constant level. In PSAPP transgenic mice, CLAC was positive in the diffuse Abeta deposits surrounding huge-cored plaques. Thus, CLAC and Abeta40 or thioS exhibit mostly separate distribution patterns in SP, suggesting that CLAC is a relatively early component of SP in human brains that may have inhibitory effects against the maturation of SP into beta-sheet-rich amyloid deposits.

CLAC: a novel Alzheimer amyloid plaque component derived from a transmembrane precursor, CLAC-P/collagen type XXV.

We raised monoclonal antibodies against senile plaque (SP) amyloid and obtained a clone 9D2, which labeled amyloid fibrils in SPs and reacted with approximately 50/100 kDa polypeptides in Alzheimer's disease (AD) brains. We purified the 9D2 antigens and cloned a cDNA encoding its precursor, which was a novel type II transmembrane protein specifically expressed in neurons. This precursor harbored three collagen-like Gly-X-Y repeat motifs and was partially homologous to collagen type XIII. Thus, we named the 9D2 antigen as CLAC (collagen-like Alzheimer amyloid plaque component), and its precursor as CLAC-P/collagen type XXV. The extracellular domain of CLAC-P/collagen type XXV was secreted by furin convertase, and the N-terminus of CLAC deposited in AD brains was pyroglutamate modified. Both secreted and membrane-tethered forms of CLAC-P/collagen type XXV specifically bound to fibrillized Abeta, implicating these proteins in beta-amyloidogenesis and neuronal degeneration in AD.