Ras-like Gem GTPase induced by Npas4 promotes activity-dependent neuronal tolerance for ischemic stroke.

Ischemic stroke, which results in loss of neurological function, initiates a complex cascade of pathological events in the brain, largely driven by excitotoxic Ca2+ influx in neurons. This leads to cortical spreading depolarization, which induces expression of genes involved in both neuronal death and survival; yet, the functions of these genes remain poorly understood. Here, we profiled gene expression changes that are common to ischemia (modeled by middle cerebral artery occlusion [MCAO]) and to experience-dependent activation (modeled by exposure to an enriched environment [EE]), which also induces Ca2+ transients that trigger transcriptional programs. We found that the activity-dependent transcription factor Npas4 was up-regulated under MCAO and EE conditions and that transient activation of cortical neurons in the healthy brain by the EE decreased cell death after stroke. Furthermore, both MCAO in vivo and oxygen-glucose deprivation in vitro revealed that Npas4 is necessary and sufficient for neuroprotection. We also found that this protection involves the inhibition of L-type voltage-gated Ca2+ channels (VGCCs). Next, our systematic search for Npas4-downstream genes identified Gem, which encodes a Ras-related small GTPase that mediates neuroprotective effects of Npas4. Gem suppresses the membrane localization of L-type VGCCs to inhibit excess Ca2+ influx, thereby protecting neurons from excitotoxic death after in vitro and in vivo ischemia. Collectively, our findings indicate that Gem expression via Npas4 is necessary and sufficient to promote neuroprotection in the injured brain. Importantly, Gem is also induced in human cerebral organoids cultured under an ischemic condition, revealing Gem as a new target for drug discovery.

Impaired Hippocampal Long-Term Potentiation and Memory Deficits upon Haploinsufficiency of MDGA1 Can Be Rescued by Acute Administration of D-Cycloserine.

The maintenance of proper brain function relies heavily on the balance of excitatory and inhibitory neural circuits, governed in part by synaptic adhesion molecules. Among these, MDGA1 (MAM domain-containing glycosylphosphatidylinositol anchor 1) acts as a suppressor of synapse formation by interfering with Neuroligin-mediated interactions, crucial for maintaining the excitatory-inhibitory (E/I) balance. Mdga1-/- mice exhibit selectively enhanced inhibitory synapse formation in their hippocampal pyramidal neurons, leading to impaired hippocampal long-term potentiation (LTP) and hippocampus-dependent learning and memory function; however, it has not been fully investigated yet if the reduction in MDGA1 protein levels would alter brain function. Here, we examined the behavioral and synaptic consequences of reduced MDGA1 protein levels in Mdga1+/- mice. As observed in Mdga1-/- mice, Mdga1+/- mice exhibited significant deficits in hippocampus-dependent learning and memory tasks, such as the Morris water maze and contextual fear-conditioning tests, along with a significant deficit in the long-term potentiation (LTP) in hippocampal Schaffer collateral CA1 synapses. The acute administration of D-cycloserine, a co-agonist of NMDAR (N-methyl-d-aspartate receptor), significantly ameliorated memory impairments and restored LTP deficits specifically in Mdga1+/- mice, while having no such effect on Mdga1-/- mice. These results highlight the critical role of MDGA1 in regulating inhibitory synapse formation and maintaining the E/I balance for proper cognitive function. These findings may also suggest potential therapeutic strategies targeting the E/I imbalance to alleviate cognitive deficits associated with neuropsychiatric disorders.

Amyloidogenic processing of amyloid ß protein precursor (APP) is enhanced in the brains of alcadein α-deficient mice.

Alzheimer's disease (AD) is a very common neurodegenerative disorder, chiefly caused by increased production of neurotoxic ß-amyloid (Aß) peptide generated from proteolytic cleavage of ß-amyloid protein precursor (APP). Except for familial AD arising from mutations in the APP and presenilin (PSEN) genes, the molecular mechanisms regulating the amyloidogenic processing of APP are largely unclear. Alcadein α/calsyntenin1 (ALCα/CLSTN1) is a neuronal type I transmembrane protein that forms a complex with APP, mediated by the neuronal adaptor protein X11-like (X11L or MINT2). Formation of the ALCα-X11L-APP tripartite complex suppresses Aß generation in vitro, and X11L-deficient mice exhibit enhanced amyloidogenic processing of endogenous APP. However, the role of ALCα in APP metabolism in vivo remains unclear. Here, by generating ALCα-deficient mice and using immunohistochemistry, immunoblotting, and co-immunoprecipitation analyses, we verified the role of ALCα in the suppression of amyloidogenic processing of endogenous APP in vivo We observed that ALCα deficiency attenuates the association of X11L with APP, significantly enhances amyloidogenic ß-site cleavage of APP, especially in endosomes, and increases the generation of endogenous Aß in the brain. Furthermore, we noted amyloid plaque formation in the brains of human APP-transgenic mice in an ALCα-deficient background. These results unveil a potential role of ALCα in protecting cerebral neurons from Aß-dependent pathogenicity in AD.

Cytoplasmic fragment of Alcadein α generated by regulated intramembrane proteolysis enhances amyloid ß-protein precursor (APP) transport into the late secretory pathway and facilitates APP cleavage.

The neural type I membrane protein Alcadein α (Alcα), is primarily cleaved by amyloid ß-protein precursor (APP) α-secretase to generate a membrane-associated carboxyl-terminal fragment (Alcα CTF), which is further cleaved by γ-secretase to secrete p3-Alcα peptides and generate an intracellular cytoplasmic domain fragment (Alcα ICD) in the late secretory pathway. By association with the neural adaptor protein X11L (X11-like), Alcα and APP form a ternary complex that suppresses the cleavage of both Alcα and APP by regulating the transport of these membrane proteins into the late secretory pathway where secretases are active. However, it has not been revealed how Alcα and APP are directed from the ternary complex formed largely in the Golgi into the late secretory pathway to reach a nerve terminus. Using a novel transgenic mouse line expressing excess amounts of human Alcα CTF (hAlcα CTF) in neurons, we found that expression of hAlcα CTF induced excess production of hAlcα ICD, which facilitated APP transport into the nerve terminus and enhanced APP metabolism, including Aß generation. In vitro cell studies also demonstrated that excess expression of Alcα ICD released both APP and Alcα from the ternary complex. These results indicate that regulated intramembrane proteolysis of Alcα by γ-secretase regulates APP trafficking and the production of Aß in vivo.

d-Allose Attenuates Overexpression of Inflammatory Cytokines after Cerebral Ischemia/Reperfusion Injury in Gerbil.

BACKGROUND: The present study investigates the effects of d-allose, a rare sugar, on the inflammatory response after transient forebrain ischemia in the gerbil and whether it reduces oxidative stress (8-hydroxyl-2'-deoxyguanosine levels) and behavioral deficits. METHODS: Transient forebrain ischemia was induced by occlusion of the bilateral common carotid arteries for 5 minutes. d-Allose was intraperitoneally injected immediately after ischemia (400 mg/kg). Inflammatory cytokines and oxidative damage in the hippocampus and behavioral deficits were examined 3 days after ischemia. RESULTS: d-Allose administration reduced ischemia-induced cytokine production, oxidative stress, and behavioral deficits (motor and memory related). CONCLUSIONS: The present results suggest that d-allose reduces brain injury after transient global ischemia by suppressing inflammation as well as by inhibiting oxidative stress.

A small peptide sequence is sufficient for initiating kinesin-1 activation through part of TPR region of KLC1.

Kinesin-1 anterogradely transports vesicles containing cargo proteins when a protein-protein interaction activates it from an inhibited state. The C-terminal cytoplasmic region of kinesin-1 cargo protein Alcadeinα (Alcα) interacts with the KLC1 subunit's tetratricopeptide repeat (TPR) region, activating kinesin-1's association with vesicles and anterograde transport. We found that either of two 10-amino-acid WD motifs in Alcα cytoplasmic region was necessary and sufficient to initiate this activation. An artificial transmembrane protein containing either WD motif induced kinesin-1's vesicular association and anterograde transport in a KLC-dependent manner, even in the normally inhibiting presence of excess KLC1, thus allowing us to analyze the KLC1 TPR-WD functional interaction in detail in vivo. A part of TPR region was dispensable for the WD motifs' activation of kinesin-1 and transport, indicating that only part of the TPR structure is required for this function in vivo. For a different kinesin-1 cargo protein, JIP1, an 11-amino-acid C-terminal region was sufficient to recruit KLC1 to vesicles, but did not activate transport. These observations suggest that structurally different TPR-interacting peptides may have different effects on kinesin-1. This mechanism may partly explain how kinesin-1 can organize the transport of a wide variety of cargo molecules.

Suppression of the amyloidogenic metabolism of APP and the accumulation of Aß by alcadein α in the brain during aging.

Generation and accumulation of amyloid-ß (Aß) protein in the brain are the primary causes of Alzheimer's disease (AD). Alcadeins (Alcs composed of Alcα, Alcß and Alcγ family) are a neuronal membrane protein that is subject to proteolytic processing, as is Aß protein precursor (APP), by APP secretases. Previous observations suggest that Alcs are involved in the pathophysiology of Alzheimer's disease (AD). Here, we generated new mouse AppNL-F (APP-KI) lines with either Alcα- or Alcß-deficient background and analyzed APP processing and Aß accumulation through the aging process. The Alcα-deficient APP-KI (APP-KI/Alcα-KO) mice enhanced brain Aß accumulation along with increased amyloidogenic ß-site cleavage of APP through the aging process whereas Alcß-deficient APP-KI (APP-KI/Alcß-KO) mice neither affected APP metabolism nor Aß accumulation at any age. More colocalization of APP and BACE1 was observed in the endolysosomal pathway in neurons of APP-KI/Alcα-KO mice compared to APP-KI and APP-KI/Alcß-KO mice. These results indicate that Alcα plays an important role in the neuroprotective function by suppressing the amyloidogenic cleavage of APP by BACE1 in the brain, which is distinct from the neuroprotective function of Alcß, in which p3-Alcß peptides derived from Alcß restores the viability in neurons impaired by toxic Aß.

Large-scale animal model study uncovers altered brain pH and lactate levels as a transdiagnostic endophenotype of neuropsychiatric disorders involving cognitive impairment.

Increased levels of lactate, an end-product of glycolysis, have been proposed as a potential surrogate marker for metabolic changes during neuronal excitation. These changes in lactate levels can result in decreased brain pH, which has been implicated in patients with various neuropsychiatric disorders. We previously demonstrated that such alterations are commonly observed in five mouse models of schizophrenia, bipolar disorder, and autism, suggesting a shared endophenotype among these disorders rather than mere artifacts due to medications or agonal state. However, there is still limited research on this phenomenon in animal models, leaving its generality across other disease animal models uncertain. Moreover, the association between changes in brain lactate levels and specific behavioral abnormalities remains unclear. To address these gaps, the International Brain pH Project Consortium investigated brain pH and lactate levels in 109 strains/conditions of 2294 animals with genetic and other experimental manipulations relevant to neuropsychiatric disorders. Systematic analysis revealed that decreased brain pH and increased lactate levels were common features observed in multiple models of depression, epilepsy, Alzheimer's disease, and some additional schizophrenia models. While certain autism models also exhibited decreased pH and increased lactate levels, others showed the opposite pattern, potentially reflecting subpopulations within the autism spectrum. Furthermore, utilizing large-scale behavioral test battery, a multivariate cross-validated prediction analysis demonstrated that poor working memory performance was predominantly associated with increased brain lactate levels. Importantly, this association was confirmed in an independent cohort of animal models. Collectively, these findings suggest that altered brain pH and lactate levels, which could be attributed to dysregulated excitation/inhibition balance, may serve as transdiagnostic endophenotypes of debilitating neuropsychiatric disorders characterized by cognitive impairment, irrespective of their beneficial or detrimental nature.

Membrane-microdomain localization of amyloid ß-precursor protein (APP) C-terminal fragments is regulated by phosphorylation of the cytoplasmic Thr668 residue.

Amyloid ß-precursor protein (APP) is primarily cleaved by α- or ß-secretase to generate membrane-bound, C-terminal fragments (CTFs). In turn, CTFs are potentially subject to a second, intramembrane cleavage by γ-secretase, which is active in a lipid raft-like membrane microdomain. Mature APP (N- and O-glycosylated APP), the actual substrate of these secretases, is phosphorylated at the cytoplasmic residue Thr(668) and this phosphorylation changes the overall conformation of the cytoplasmic domain of APP. We found that phosphorylated and nonphosphorylated CTFs exist equally in mouse brain and are kinetically equivalent as substrates for γ-secretase, in vitro. However, in vivo, the level of the phosphorylated APP intracellular domain peptide (pAICD) generated by γ-cleavage of CTFs was very low when compared with the level of nonphosphorylated AICD (nAICD). Phosphorylated CTFs (pCTFs), rather than nonphosphorylated CTFs (nCTFs), were preferentially located outside of detergent-resistant, lipid raft-like membrane microdomains. The APP cytoplasmic domain peptide (APP(648-695)) with Thr(P)(668) did not associate with liposomes composed of membrane lipids from mouse brain to which the nonphosphorylated peptide preferentially bound. In addition, APP lacking the C-terminal 8 amino acids (APP-ΔC8), which are essential for membrane association, decreased Aß generation in N2a cells. These observations suggest that the pCTFs and CTFΔC8 are relatively movable within the membrane, whereas the nCTFs are susceptible to being anchored into the membrane, an interaction made available as a consequence of not being phosphorylated. By this mechanism, nCTFs can be preferentially captured and cleaved by γ-secretase. Preservation of the phosphorylated state of APP-CTFs may be a potential treatment to lower the generation of Aß in Alzheimer disease.

Molecular mechanisms underlying activity-dependent ischemic tolerance in the brain.

Ischemic stroke is one of the leading causes of death and disability worldwide. The inhibition of cerebral blood flow triggers intertwined pathological events, resulting in cell death and loss of brain function. Interestingly, animals pre-exposed to short-term ischemia can tolerate subsequent severe ischemia. This phenomenon is called ischemic tolerance and is also triggered by other noxious stimuli. However, whether short-term exposure to non-noxious stimuli can induce ischemic tolerance remains unknown. Recently, we found that pre-exposing mice to an enriched environment for 40 min is sufficient to facilitate cell survival after a subsequent stroke. The neuroprotective process depends on the neuronal activity soon before stroke, of which the activity-dependent transcription factor Npas4 is essential. Excessive Ca2+ influx triggers Npas4 expression in ischemic neurons, leading to the activation of neuroprotective programs. Pre-induction of Npas4 in the normal brain effectively supports cell survival after stroke. Furthermore, our study revealed that Npas4 regulates L-type voltage-gated Ca2+ channels through expression of the small Ras-like GTPase Gem in ischemic neurons. Ischemic tolerance is a good model for understanding how to promote neuroprotective mechanisms in the normal and injured brain. Here, we highlight activity-dependent ischemic tolerance and discuss its role in promoting neuroprotection against stroke.

Early postnatal development of the MDGA2+/- mouse model of synaptic dysfunction.

Synaptic dysfunction underlies many neurodevelopmental disorders (NDDs). The membrane-associated mucin domain-containing glycosylphosphatidylinositol anchor proteins (MDGAs) regulate synaptic development by modulating neurexin-neuroligin complex formation. Since understanding the neurodevelopmental profile and the sex-based differences in the manifestation of the symptoms of NDDs is important for their early diagnosis, we tested a mouse model haploinsufficient for MDGA2 (MDGA2+/-) on a neurodevelopmental test battery, containing sensory, motor, and cognitive measures, as well as ultrasonic vocalizations. When male and female MDGA2+/- and wildtype (WT) C57BL/6 J mice were examined from 2 to 23 days of age using this test battery, genotype and sex differences in body weight, sensory-motor processes, and ultrasonic vocalizations were observed. The auditory startle reflex appeared earlier in the MDGA2+/- than in WT mice and the MDGA2+/- mice produced fewer ultrasonic vocalizations. The MDGA2+/- mice showed reduced locomotion and rearing than WT mice in the open field after 17 days of age and spent less time investigating a novel object than WT mice at 21 days of age. Female MDGA2+/- mice weighed less than WT females and showed lower grip strength, indicating a delay in sensory-motor development in MDGA2+/- mice, which appears to be more pronounced in females than males. The behavioural phenotypes resulting from MDGA2 haploinsufficiency suggests that it shows delayed development of motor behaviour, grip strength and exploratory behaviour, non-social phenotypes of NDDs.

Axonal transport of Frizzled5 by Alcadein α-containing vesicles is associated with kinesin-1.

Alcadein α (Alcα) and amyloid-ß protein precursor (APP) are cargo receptors that associate vesicles with kinesin-1. These vesicles, which contain either Alcα or APP, transport various proteins/cargo molecules into axon nerve terminals. Here, we analyzed immune-isolated Alcα- and APP-containing vesicles of adult mouse brains with LC-MS/MS and identified proteins present in vesicles that contained either Alcα or APP. Among these proteins, Frizzled-5 (Fzd5), a Wnt receptor, was detected mainly in Alcα vesicles. Although colocalization ratios of Fzd5 with Alcα are low in the neurites of differentiating neurons by a low expression of Fzd5 in embryonic brains, the suppression of Alcα expression decreased the localization of Fzd5 in neurites of primary cultured neurons. Furthermore, Fzd5-EGFP expressed in primary cultured neurons was preferentially transported in axons with the transport velocities of Alcα vesicles. In synaptosomal fractions of adult-mice brains that express higher levels of Fzd5, the amount of Fzd5 and the phosphorylation level of calcium/calmodulin-dependent protein kinase-II were reduced in the Alcα-deficient mice. These results suggest that reduced transport of Fzd5 by Alcα-containing vesicles associated with kinesin-1 in axon terminals may impair the response to Wnt ligands in the noncanonical Ca2+-dependent signal transduction pathway at nerve terminals of mature neurons.

Brain p3-Alcß peptide restores neuronal viability impaired by Alzheimer's amyloid ß-peptide.

We propose a new therapeutic strategy for Alzheimer's disease (AD). Brain peptide p3-Alcß37 is generated from the neuronal protein alcadein ß through cleavage of γ-secretase, similar to the generation of amyloid ß (Aß) derived from Aß-protein precursor/APP. Neurotoxicity by Aß oligomers (Aßo) is the prime cause prior to the loss of brain function in AD. We found that p3-Alcß37 and its shorter peptide p3-Alcß9-19 enhanced the mitochondrial activity of neurons and protected neurons against Aßo-induced toxicity. This is due to the suppression of the Aßo-mediated excessive Ca2+ influx into neurons by p3-Alcß. Successful transfer of p3-Alcß9-19 into the brain following peripheral administration improved the mitochondrial viability in the brain of AD mice model, in which the mitochondrial activity is attenuated by increasing the neurotoxic human Aß42 burden, as revealed through brain PET imaging to monitor mitochondrial function. Because mitochondrial dysfunction is common in the brain of AD patients alongside increased Aß and reduced p3-Alcß37 levels, the administration of p3-Alcß9-19 may be a promising treatment for restoring, protecting, and promoting brain functions in patients with AD.

Alternative processing of γ-secretase substrates in common forms of mild cognitive impairment and Alzheimer's disease: evidence for γ-secretase dysfunction.

OBJECTIVE: The most common pathogenesis for familial Alzheimer's disease (FAD) involves misprocessing (or alternative processing) of the amyloid precursor protein (APP) by γ-secretase due to mutations of the presenilin 1 (PS1) gene. This misprocessing/alternative processing leads to an increase in the ratio of the level of a minor γ-secretase reaction product (Aß42) to that of the major reaction product (Aß40). Although no PS1 mutations are present, altered Aß42/40 ratios are also observed in sporadic Alzheimer's disease (SAD), and these altered ratios apparently reflect deposition of Aß42 as amyloid. METHODS: Using immunoprecipitation-mass spectrometry with quantitative accuracy, we analyzed in the cerebrospinal fluid (CSF) of various clinical populations the peptide products generated by processing of not only APP but also an unrelated protein, alcadein (Alc). Alc undergoes metabolism by the identical APP α-secretases and γ-secretases, yielding a fragment that we have named p3-Alc(α) because of the parallel genesis of p3-Alc(α) peptides and the p3 fragment of APP. As with Aß, both major and minor p3-Alc(α) s are generated. We studied the alternative processing of p3-Alc(α) in various clinical populations. RESULTS: We previously reported that changes in the Aß42/40 ratio showed covariance in a linear relationship with the levels of p3-Alc(α) [minor/major] ratio in media conditioned by cells expressing FAD-linked PS1 mutants. Here we studied the speciation of p3-Alc(α) in the CSF from 3 groups of human subjects (n = 158): elderly nondemented control subjects; mild cognitive impairment (MCI) subjects with a clinical dementia rating (CDR) of 0.5; SAD subjects with CDR of 1.0; and other neurological disease (OND) control subjects. The CSF minor p3-Alc(α) variant, p3-Alc(α) 38, was elevated (p < 0.05) in MCI subjects or SAD subjects, depending upon whether the data were pooled and analyzed as a single cohort or analyzed individually as 3 separate cohorts. INTERPRETATION: These results suggest that some SAD may involve alternative processing of multiple γ-secretase substrates, raising the possibility that the molecular pathogenesis of SAD might involve γ-secretase dysfunction.

IgSF molecule MDGA1 is involved in radial migration and positioning of a subset of cortical upper-layer neurons.

Mdga1, encoding a GPI-anchored immunoglobulin superfamily molecule containing an MAM domain, is expressed by a specific subset of neurons, including layer II/III projection neurons, in the mouse neocortex. To investigate the function of Mdga1 in corticogenesis, we generated Mdga1-deficient mice and backcrossed them to obtain a congenic background. Gross anatomy of the Mdga1-deficient brain at postnatal day (P) 14 showed no obvious phenotype. However, the migration of Mdga1-mutant neurons to the superficial cortical plate was clearly delayed. Most Mdga1-mutant neurons reached the lower portion of the upper cortical layer by embryonic day 18.5 and stayed there through P0. By P7, the location of the mutant cells was the same as wild-type. The location of Cux2-expressing upper-layer neurons in the cortical plate was largely unaffected. These observations indicated that Mdga1 is involved in the migration and positioning of a subset of cortical neurons and suggested that the radial migration of upper-layer neurons might be differentially regulated.

Formation of N-acyl-phosphatidylethanolamines by cytosolic phospholipase A2ε in an ex vivo murine model of brain ischemia.

N-Acyl-phosphatidylethanolamines (NAPEs), a minor class of membrane glycerophospholipids, accumulate along with their bioactive metabolites, N-acylethanolamines (NAEs) during ischemia. NAPEs can be formed through N-acylation of phosphatidylethanolamine by cytosolic phospholipase A2ε (cPLA2ε, also known as PLA2G4E) or members of the phospholipase A and acyltransferase (PLAAT) family. However, the enzyme responsible for the NAPE production in brain ischemia has not yet been clarified. Here, we investigated a possible role of cPLA2ε using cPLA2ε-deficient (Pla2g4e-/-) mice. As analyzed with brain homogenates of wild-type mice, the age dependency of Ca2+-dependent NAPE-forming activity showed a bell-shape pattern being the highest at the first week of postnatal life, and the activity was completely abolished in Pla2g4e-/- mice. However, liquid chromatography-tandem mass spectrometry revealed that the NAPE levels of normal brain were similar between wild-type and Pla2g4e-/- mice. In contrast, post-mortal accumulations of NAPEs and most species of NAEs were only observed in decapitated brains of wild-type mice. These results suggested that cPLA2ε is responsible for Ca2+-dependent formation of NAPEs in the brain as well as the accumulation of NAPEs and NAEs during ischemia, while other enzyme(s) appeared to be involved in the maintenance of basal NAPE levels.

Ascending spinal tract formation in chick embryo originating from different spinal regions.

The present study aimed to assess spinal tract formation in neurons originating from cervical (C7), brachial (C14), and thoracic (T4) regions, with the lumbar (LS2) region as a reference, in a chick embryo. For the assessment of the spinal tracts, we introduced a vector expressing human placental alkaline phosphatase into progenitor cells generated after neural tube closure and belonging to the above segments, using in ovo electroporation. The ascending axons took primarily similar paths: dorsal commissural, ventral commissural, and dorsal non-commissural paths, with some variance depending on their originating segments. Some populations of non-commissural neurons later extended their axons following a ventral path. The elongation rates of these axons are primarily constant and tended to increase over time; however, some variations depending on the originating segments were also observed. Some of the dorsally ascending axons entered into the developing cerebellum, and spinocerebellar neurons originating from T4 projected their axons into the cortex of the cerebellum differently from those from LS2. These results unveil an overall picture of early ascending spinal tract formation.

Alcadein cleavages by amyloid beta-precursor protein (APP) alpha- and gamma-secretases generate small peptides, p3-Alcs, indicating Alzheimer disease-related gamma-secretase dysfunction.

Alcadeins (Alcs) constitute a family of neuronal type I membrane proteins, designated Alc(alpha), Alc(beta), and Alc(gamma). The Alcs express in neurons dominantly and largely colocalize with the Alzheimer amyloid precursor protein (APP) in the brain. Alcs and APP show an identical function as a cargo receptor of kinesin-1. Moreover, proteolytic processing of Alc proteins appears highly similar to that of APP. We found that APP alpha-secretases ADAM 10 and ADAM 17 primarily cleave Alc proteins and trigger the subsequent secondary intramembranous cleavage of Alc C-terminal fragments by a presenilin-dependent gamma-secretase complex, thereby generating "APP p3-like" and non-aggregative Alc peptides (p3-Alcs). We determined the complete amino acid sequence of p3-Alc(alpha), p3-Alc(beta), and p3-Alc(gamma), whose major species comprise 35, 37, and 31 amino acids, respectively, in human cerebrospinal fluid. We demonstrate here that variant p3-Alc C termini are modulated by FAD-linked presenilin 1 mutations increasing minor beta-amyloid species Abeta42, and these mutations alter the level of minor p3-Alc species. However, the magnitudes of C-terminal alteration of p3-Alc(alpha), p3-Alc(beta), and p3-Alc(gamma) were not equivalent, suggesting that one type of gamma-secretase dysfunction does not appear in the phenotype equivalently in the cleavage of type I membrane proteins. Because these C-terminal alterations are detectable in human cerebrospinal fluid, the use of a substrate panel, including Alcs and APP, may be effective to detect gamma-secretase dysfunction in the prepathogenic state of Alzheimer disease subjects.

Retinal ganglion cell loss in kinesin-1 cargo Alcadein α deficient mice.

Maintenance of retinal ganglion cells (RGCs) activity is relied on axonal transport conveying materials required for their survival such as neurotrophic factors. Kinesin-1 undergoes anterograde transport in axons, and Alcadein α (Alcα; also called calsyntenin-1) is a major cargo adaptor protein that can drive kinesin-1 to transport vesicles containing Alcα. The long-term effects of Alcα-deficiency on retinal morphology and survival of RGCs during postnatal development were examined in Alcα knockout mice. At 1.5, 3, 6, and 15 months postnatal, the number of retrogradely labeled RGCs was determined in flat-mounted retinas of Alcα-deficient and wild-type mice. Retinal damage was assessed histologically by determining the retinal thickness. Intraocular pressure (IOP) was measured with a Tonolab tonometer. At 1.5 months postnatal, the number of retrogradely labeled RGCs was not different between wild-type and Alcα-deficient mice. However, at 3, 6, and 15 months postnatal, the number of RGCs was significantly lower in Alcα deficient mice than those of wild-type mice (143 ± 41.1 cells/mm2 vs. 208 ± 28.4 cells/mm2, respectively, at 3 months; P < 0.01). No differences were seen in retinal thickness or IOP between the two types of mice at any postnatal age. Alcα-deficient mice showed spontaneous loss of RGCs but no elevation in IOP. These mice mimic normal-tension glaucoma and will be useful for investigating the mechanism of neurodegeneration in this disorder and for developing treatments for RGC loss that does not involve changes in IOP.

Visually cued fear conditioning test for memory impairment related to cortical function.

AIM: Fear conditioning tests are intended to elucidate a subject's ability to associate a conditioned stimulus with an aversive, unconditioned stimulus, such as footshock. Among these tests, a paradigm related to precise cortical functions would be increasingly important in drug screening for disorders such as schizophrenia and dementia. Therefore, we established a new fear conditioning paradigm using a visual cue in mice. In addition, the validity of the test was evaluated using a genetically engineered mouse, heterozygous deficient in Mdga1 (Mdga1+/-), which is related to schizophrenia. RESULTS: Mice were given footshocks associated with a visual cue of moving gratings at training in 25-minute sessions. The mice showed the conditioned response of freezing behavior to the visual stimulus at testing 24 hours after the footshocks. In the test for validation, the Mdga1+/- deficient mice showed significantly less freezing than wild-type mice. CONCLUSION: The visually cued fear conditioning paradigm with moving gratings has been established, which is experimentally useful to evaluate animal cortical functions. The validity of the test was confirmed for Mdga1-deficient mice with possible deficiency in cortical functions.