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Macrophage migration inhibitory factor (MIF) is involved in the function of both the innate and adaptive immune systems and in neuroprotection and has recently been implicated in multiple sclerosis (MS).Determination of MIF levels in the cerebrospinal fluid (CSF) of patients with distinct subtypes of MS and the cellular localization of MIF in human brain tissue.The levels of MIF were investigated in CSF from patients with clinically isolated syndrome (CIS) (n = 26), relapsing-remitting MS (RRMS) (n = 22), secondary progressive MS (SPMS) (n = 19), and healthy controls (HCs) (n = 24), using ELISA. The effect of disease-modifying therapies in the RRMS and SPMS cohorts were examined. Cellular distribution of MIF in the human brain was studied using immunochemistry and the newly available OligoInternode database.MIF was significantly decreased in treatment-naïve CIS and RRMS patients compared to HCs but was elevated in SPMS. Interestingly, MIF levels were sex-dependent and significantly lower in women with CIS and RRMS. MIF expression in the human brain was localized to neurons, astrocytes, pericytes, and oligo5 oligodendrocytes but not in microglia.The finding that MIF was decreased in newly diagnosed CIS and RRMS patients but was high in patients with SPMS may suggest that MIF levels in CSF are regulated by local MIF receptor expression that affects the overall MIF signaling in the brain and may represent a protective mechanism that eventually fails.

BACKGROUND: High Temperature Requirement Serine Protease A1 (HTRA1) degrades extracellular matrix molecules (ECMs) and growth factors. It interacts with several proteins implicated in multiple sclerosis (MS), but has not previously been linked to the disease.OBJECTIVE: Investigate the levels of HTRA1 in cerebrospinal fluid (CSF) in different subtypes of MS and brain tissue.METHODS: Using ELISA, HTRA1 levels were compared in CSF from untreated patients with relapsing-remitting MS (RRMS, n = 23), secondary progressive MS (SPMS, n = 26) and healthy controls (HCs, n = 26). The effect of disease modifying therapies (DMTs) were examined in both patient groups. Cellular distribution in human brain was studied using immunochemistry and the oligointernode database, based on a single-nuclei RNA expression map.RESULTS: HTRA1 increased in RRMS and SPMS compared to HCs. DMT decreased HTRA1 levels in both types of MS. Using ROC analysis, HTRA1 cut-offs could discriminate HCs from RRMS patients with 100% specificity and 82.6% sensitivity. In the brain, HTRA1 was expressed in glia and neurons.CONCLUSION: HTRA1 is a promising CSF biomarker for MS correlating with disease- and disability progression. Most cell species of the normal and diseased CNS express HTRA1 and the expression pattern could reflect pathological processes involved in MS pathogenesis.

Posttraumatic syringomyelia (PTS) is a serious condition of progressive expansion of spinal cord cysts, affecting patients with spinal cord injury years after injury. To evaluate neural cell therapy to prevent cyst expansion and potentially replace lost neurons, we developed a rat model of PTS. We combined contusive trauma with subarachnoid injections of blood, causing tethering of the spinal cord to the surrounding vertebrae, resulting in chronically expanding cysts. The cysts were usually located rostral to the injury, extracanalicular, lined by astrocytes. T2*-weighted magnetic resonance imaging (MRI) showed hyperintense fluid-filled cysts but also hypointense signals from debris and iron-laden macrophages/microglia. Two types of human neural stem/progenitor cells—fetal neural precursor cells (hNPCs) and neuroepithelial-like stem cells (hNESCs) derived from induced pluripotent stem cells—were transplanted to PTS cysts. Cells transplanted into cysts 10 weeks after injury survived at least 10 weeks, migrated into the surrounding parenchyma, but did not differentiate during this period. The cysts were partially obliterated by the cells, and cyst walls often merged with thin layers of cells in between. Cyst volume measurements with MRI showed that the volumes continued to expand in sham-transplanted rats by 102%, while the cyst expansion was effectively prevented by hNPCs and hNESCs transplantation, reducing the cyst volumes by 18.8% and 46.8%, respectively. The volume reductions far exceeded the volume of the added human cells. Thus, in an animal model closely mimicking the clinical situation, we provide proof-of-principle that transplantation of human neural stem/progenitor cells can be used as treatment for PTS. Supplementary Information The online version contains supplementary material available at 10.1007/s13311-020-00987-3.

The aim of this study was to translate and culturally adapt the Short Musculoskeletal Function Assessment (SMFA) into Danish (SMFA-DK) and assess the psychometric properties. SMFA was translated and cross-culturally adapted according to a standardized procedure. Minor changes in the wording in three items were made to adapt to Danish conditions. Acute patients (n = 201) and rehabilitation patients (n = 231) with musculoskeletal problems aged 18–87 years were included. The following analysis were made to evaluate psychometric quality of SMFA-DK: Reliability with Chronbach’s alpha, content validity as coding according to the International Classification of Functioning, Disability and Health (ICF), floor/ceiling effects, construct validity as factor analysis, correlations between SMFA-DK and Short Form 36 and also known group method. Responsiveness and effect size were calculated. Cronbach’s alpha values were between 0.79 and 0.94. SMFA-DK captured all components of the ICF, and there were no floor/ceiling effects. Factor analysis demonstrated four subscales. SMFA-DK correlated good with the SF-36 subscales for the rehabilitation patients and lower for the newly injured patients. Effect sizes were excellent and better for SMFA-DK than for SF-36. The study indicates that SMFA-DK can be a valid and responsive measure of outcome in rehabilitation settings.

Nonpluripotent neural progenitor cells (NPCs) derived from the human fetal central nervous system were found to express a number of messenger RNA (mRNA) species associated with pluripotency, such as NANOG, REX1, and OCT4. The expression was restricted to small subpopulations of NPCs. In contrast to pluripotent stem cells, there was no coexpression of the pluripotency-associated genes studied. Although the expression of these genes rapidly declined during the in vitro differentiation of NPCs, we found no evidence that the discrete expression was associated with the markers of multipotent neural stem cells (CD133+/CD24lo), the capacity of sphere formation, or high cell proliferation rates. The rate of cell death among NPCs expressing pluripotency-associated genes was also similar to that of other NPCs. Live cell imaging showed that NANOG- and REX1-expressing NPCs continuously changed morphology, as did the nonexpressing cells. Depletion experiments showed that after the complete removal of the subpopulations of NANOG- and REX1-expressing NPCs, the expression of these genes appeared in other NPCs within a few days. The percentage of NANOG- and REX1-expressing cells returned to that observed before depletion. Our results are best explained by a model in which there is stochastic transient expression of pluripotency-associated genes in proliferating NPCs.

Macrophage migration inhibitory factor (MIF), a small conserved protein, is abundant in the immune- and central nervous system (CNS). MIF has several receptors and binding partners that can modulate its action on a cellular level. It is upregulated in neurodegenerative diseases and cancer although its function is far from clear. Here, we report the finding of a new binding partner to MIF, the serine protease HTRA1. This enzyme cleaves several growth factors, extracellular matrix molecules and is implicated in some of the same diseases as MIF. We show that the function of the binding between MIF and HTRA1 is to inhibit the proteolytic activity of HTRA1, modulating the availability of molecules that can change cell growth and differentiation. MIF is therefore the first endogenous inhibitor ever found for HTRA1. It was found that both molecules were present in astrocytes and that the functional binding has the ability to modulate astrocytic activities important in development and disease of the CNS. Electronic supplementary material The online version of this article (doi:10.1007/s00018-017-2592-z) contains supplementary material, which is available to authorized users.

Incorporation of the N-methyl-d-aspartate receptor (NMDAR) subunit NR3A into functional NMDARs results in reduced channel conductance and Ca2+ permeability. To further investigate the function of NR3A, we have set out to characterize its intracellular binding partners. Here, we report a novel protein interaction between NR3A and microtubule associated-protein (MAP) 1B, which both are localized to dendritic shafts and filopodia. NR3A protein levels were increased in MAP1B deficient (−/−) mice, with a corresponding decrease in NR1 levels, but the fraction of filopodia immunoreactive for NR3A was equal in cells from −/− and wild type (WT) mice. NR3A has previously been shown to interact with another member of the MAP1 family, MAP1S. We showed that MAP1S binds to microtubules in a similar manner as MAP1B, and suggest that MAP1S and MAP1B both are involved in regulating trafficking of NR3A-containing NMDAR., This work was supported by the Swedish Research Council, Marianne and Marcus Wallenberg Foundation and the Karolinska Institutet research funds.

As a disease model, the laboratory rat has contributed enormously to neuroscience research over the years. It has also been a popular animal model for Alzheimer's disease but its popularity has diminished during the last decade, as techniques for genetic manipulation in rats have lagged behind that of mice. In recent years, the rat has been making a comeback as an Alzheimer's disease model and the appearance of increasing numbers of transgenic rats will be a welcome and valuable complement to the existing mouse models. This review summarizes the contributions and current status of the rat as an animal model of Alzheimer's disease.

Altered calcium homeostasis is implicated in the pathogenesis of Alzheimer's disease (AD). Much effort has been put into understanding the association between protein mutations causative of this devastating neurodegenerative disease and perturbed calcium signaling. Whereas the presenilin mutations have received most attention in the context of neuronal calcium signaling, we focused on the effects of APP with the so-called Swedish mutation (APPswe) on spontaneous neuronal activity. We observed that primary hippocampal neurons from an APPswe transgenic rat showed increased frequency and unaltered amplitude of spontaneous calcium oscillations as compared to wild-type neurons. We found that the altered calcium signaling of APPswe transgenic neurons was unlikely to be due to modulation of the NMDA or nicotinic neurotransmitter systems, and did not depend on secreted APP derivates. The implications of this effect of APP are discussed.

NR3A, representing the third class of NMDA receptor subunits, was first studied in rats, demonstrating ubiquitous expression in the developing central nervous system (CNS), but in the adult mainly expressed in spinal cord and some forebrain nuclei. Subsequent studies showed that rodent and non-human primate NR3A expression differs. We have studied the distribution of NR3A in the human CNS and show a widespread distribution of NR3A protein in adult human brain. NR3A mRNA and protein were found in all regions of the cerebral cortex, and also in the subcortical forebrain, midbrain and hindbrain. Only very low levels of NR3A mRNA and protein could be detected in homogenized adult human spinal cord, and in situ hybridization showed that expression was limited to ventral motoneurons. We found that NR3A is associated with NR1, NR2A and NR2B in adult human CNS, suggesting the existence of native NR1-NR2A/B-NR3A assemblies in adult human CNS. While NR1 and NR2A could only be efficiently solubilized by deoxycholate, NR3A was extracted by all detergents, suggesting that a large fraction is weakly anchored to cell membranes and other proteins. Using size exclusion chromatography we found that just as for NR1, a large fraction of NR3A exists as monomers and dimers, suggesting that these two glycine binding subunits behave similarly with regard to receptor assembly and trafficking.

In the present paper we describe our on-going project investigating the functional roles of the N-methyl-D-aspartate (NMDA) receptor subunit NR3A. We find that NR3A mRNA is abundant both in embryonic and adult human brain, in contrast to the almost non-existing expression in adult rodent brain. Human NR3A (hNR3A) protein expression is particularly abundant in the cerebral cortex, as shown by western blot using NR3A-specific antibodies. Distribution of hNR3A in adult human brain shows a similar pattern as NR3A in post-natal rodent brain. We have previously reported that NR3A contains a glycine binding site, with similar affinity as the glycine binding site of NR1 subunits. This suggests that NR3A may replace one of the two NR1 subunits in native NMDA receptors. Cloning of hNR3A showed a human-specific polyproline-sequence in the intracellular C-terminus, that may bind to SH3-domains. We hypothesized that the significant differences in expression in the adult human and rodent brain could be due to an atypical interaction of hNR3A with the SH3 domain of the synaptic scaffolding protein PSD-95, that binds to NR2 subunits through its PDZ domains. However, using a number of different protein interaction assays, binding of PSD-95 to hNR3A could no be demonstrated either in vitro or in vivo. To identify intracellular signaling pathways for NR3A-containing NMDA receptors, we screened for proteins interacting with hNR3A and identified three proteins: plectin, CARP-1 and GPS2. The possible physiological roles of these interactions are discussed.

Presenilins (PSs) are involved in processing several proteins such as the amyloid precursor protein (APP), as well as in pathways for cell death and survival. We previously showed that some familial Alzheimer's disease PS mutations cause increased basal and acetylcholine muscarinic receptor-stimulated phospholipase C (PLC) activity which was gamma-secretase dependent. To further evaluate the dependence of PLC on PSs we measured PLC activity and the activation of variant protein kinase C (PKC) isoforms in mouse embryonic fibroblasts (MEFs) lacking either PS1, PS2, or both. PLC activity and PKCalpha and PKCgamma activations were significantly lower in PS1 and PS2 double knockout MEFs after PLC stimulation. Protein levels of PKCalpha and PKCgamma were lower in PS1 and PS2 double knockout MEFs. In contrast, PKCdelta levels were significantly elevated in PS1 and PS2 double knockout as well as in PS1 knockout MEFs. Also, PKCdelta levels were lowered after transfection of PS1 into PS1 knockout or PS double knockout MEFs. Using APP knockout MEFs we showed that the expression of PKCalpha, but not the other PKC isoforms is partially dependent on APP and can be regulated by APP intracellular domain (AICD). These results show that PLC and PKC activations are modulated by PS and also that PSs differentially regulate the expression of PKC isoforms by both APP/AICD-dependent and independent mechanisms.

Alzheimer’s disease is a neurodegenerative disorder characterised by extracellular accumulation of the Aβ peptide, derived from the amyloid precursor protein (APP). The function of APP as a cell surface receptor was examined by ligand-mimicking using an antibody against the APP extracellular domain. Alterations in gene expression evoked by antibody-bound APP were analysed using human pathway-finder gene arrays and the largest change in expression levels was found for ornithine decarboxylase (ODC). These results were confirmed by Western blotting which showed even higher upregulation on the protein level. APP knockdown by RNAi verified that upregulation of ODC was APP-mediated. This APP signalling event did not require γ-secretase cleavage, as it was independent of the presence of presenilin-1 or -2. The induced ODC expression was rapid and biphasic, resembling growth-factor stimulated signalling events. This study shows that antibody-bound APP leads to altered gene expression that may be relevant to AD.

Purpose of review A number of Alzheimer transgenic mouse models have been generated using a variety of different constructs containing the amyloid precursor protein. Some of the models develop features of Alzheimer's disease, including amyloid pathology, cholinergic deficits and cognitive impairment. Recent publications have demonstrated the benefits of using transgenic animals as models of Alzheimer's disease. Recent findings Genetic crosses with animals expressing other transgenes relevant to Alzheimer's disease have enhanced our understanding of the underlying pathogenic mechanisms. In the past year the Alzheimer transgenic models have increasingly been used in the development of anti-Alzheimer drugs. Summary Although the current transgenic animals are still not complete models of Alzheimer's disease, they are useful for studies on pathogenic mechanisms and the evaluation of new anti-amyloid treatment strategies.

Native N-methyl-D-aspartate (NMDA) receptors are heteromeric assemblies of four or five subunits. The NMDA receptor subunits, NR1, NR2A, NR2B, NR2C, and NR2D have been cloned in several species, including man. The NR3A subunit, which in rodents is predominantly expressed during early development, seems to function by reducing the NMDA receptor response. The human homologue to the rat NR3A, however, had not been cloned. In order to study the functions of the human NR3A (hNR3A), we have cloned and sequenced the hNR3A. It was found to share 88% of the DNA sequence with the rat gene, corresponding to a 93% homology at the amino acid level. Based on available data from human genome databases, we localized the gene to chromosome 9. The transcript could be detected by in situ hybridization in human fetal spinal cord and forebrain. Two splice variants of NR3A have been reported in rat brain, the longer of the two containing a 60 bp insert in the intracellular domain. We were unable to detect this 60 bp insert in fetal or adult human brain, suggesting that only the short variant is expressed in humans.

The γ-aminobutyric acid (GABA) type B receptor has been implicated in glial cell development in the peripheral nervous system (PNS), although the exact function of GABA signaling is not known. To investigate GABA and its B receptor in PNS development and degeneration, we studied the expression of the GABAB receptor, GABA, and glutamic acid decarboxylase GAD65/67 in both development and injury in fetal dissociated dorsal root ganglia (DRG) cell cultures and in the rat sciatic nerve. We found that GABA, GAD65/67, and the GABAB receptor were expressed in premyelinating and nonmyelinating Schwann cells throughout development and after injury. A small population of myelinated sensory fibers displayed all of these molecules at the node of Ranvier, indicating a role in axon-glia communication. Functional studies using GABAB receptor agonists and antagonists were performed in fetal DRG primary cultures to study the function of this receptor during development. The results show that GABA, via its B receptor, is involved in the myelination process but not in Schwann cell proliferation. The data from adult nerves suggest additional roles in axon-glia communication after injury.

Alzheimer's disease (AD) is characterized by the degeneration and loss of neurons, intracellular neurofibrillary tangles and the accumulation of extracellular senile plaques consisting mainly of beta-amyloid (A beta). A beta is generated from the amyloid precursor protein (APP) by sequential beta- and gamma-secretase cleavage. Alternatively, APP may be cleaved within the A beta region by alpha-secretase, preventing A beta formation. Here we investigated APP processing and secretion in primary neurons, using either colchicine or the calcium ionophore A23187 to induce apoptosis. Cell viability was determined by MTT measurements and apoptosis was further confirmed by annexin V and propidium iodide staining. We found that exposure to A23187 significantly decreased the secretion of soluble beta-secretase cleaved APP (beta-sAPP) in a caspase-dependent manner, although the secretion of total soluble APP beta sAPP) did not change. In addition, caspase inhibition restored cell viability to control levels. Exposure to colchicine did not change the amount of either secreted beta-sAPP or total sAPP and caspase inhibition was only partially able to restore cell viability. We conclude that calcium homeostasis is an important apoptotic effector specifically affecting the beta-secretase cleavage of APP.

Alternative cleavage of the amyloid precursor protein (APP) results in generation and secretion of both soluble APP (sAPP) and beta-amyloid (Abeta). Abeta is the main component of the amyloid depositions in the brains of Alzheimer's disease (AD) patients. Using Western blotting, we compared the levels of alpha-secretase cleaved sAPP, beta-secretase cleaved sAPP and total sAPP, in cerebrospinal fluid (CSF) from 13 sporadic AD patients and 13 healthy controls. Our findings show significant amounts of beta-secretase cleaved sAPP in CSF. There was no statistically significant difference in the levels of beta-secretase cleaved sAPP between AD patients and controls. The levels of alpha-secretase cleaved sAPP and total sAPP were, however, found to be significantly lower in the AD patients than in the controls.

Protein kinase C (PKC) is a multigene family of at least ten isoforms, nine of which are expressed in brain (alpha, betaI, betaII, gamma, delta, straightepsilon, eta, zeta, iota/lambda). Our previous studies have shown that many of these PKCs participate in synaptic plasticity in the CA1 region of the hippocampus. Multiple isoforms are transiently activated in the induction phase of long-term potentiation (LTP). In contrast, a single species, zeta, is persistently activated during the maintenance phase of LTP through the formation of an independent, constitutively active catalytic domain, protein kinase Mzeta (PKMzeta). In this study, we used immunoblot and immunocytochemical techniques with isoform-specific antisera to examine the distribution of the complete family of PKC isozymes and PKMzeta in rat brain. Each form of PKC showed a widespread distribution in the brain with a distinct regional pattern of high and low levels of expression. PKMzeta, the predominant form of PKM in brain, had high levels in hippocampus, frontal and occipital cortex, striatum, and hypothalamus. In the hippocampus, each isoform was expressed in a characteristic pattern, with zeta prominent in the CA1 stratum radiatum. These results suggest that the compartmentalization of PKC isoforms in neurons may contribute to their function, with the location of PKMzeta prominent in areas notable for long-term synaptic plasticity.

Mutations in the presenilin-1 (PS1) and presenilin-2 (PS2) genes account for the majority of early-onset familial Alzheimer's disease cases. Recent studies suggest that presenilin gene mutations predispose cells to apoptosis by mechanisms involving altered calcium homeostasis and oxidative damage. In the present study, we determined whether PS1 mutations also sensitize cells to hyperosmotic stress-induced apoptosis. For this, we established SH-SY5Y neuroblastoma cell lines stably transfected with wild-type PS1 or either the PS1 exon 9 deletion (deltaE9) or PS1 L250S mutants. Cultured cells were exposed to an overnight (17 h) serum deprivation, followed by a 30 min treatment with either 20 mM glucose, 10 nM insulin-like growth factor-1 or 20 mM glucose + 10 nM insulin-like growth factor-1. Cells were then cultured for a further 3, 6 or 24 h and stained for apoptotic condensed nuclei using propidium iodide. Confirmation that cells were undergoing an active apoptotic process was achieved by labelling of DNA strand breaks using the terminal dUTP nick end labelling (TUNEL) technique. We also determined cell viability using 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. Propidium iodide staining revealed that all cell lines and controls showed an increased number of apoptotic cells appearing with condensed nuclei at 24 h compared with 6 h and 3 h. High glucose-induced hyperosmotic stress resulted in significantly more apoptotic cells in the PS1 deltaE9 and PS1 L250S mutation cell lines at 24 h, compared with the wild-type PS1 lines (P

An important gap in our understanding of the pathogenesis of the amyloidoses is the identification of the cellular events that lead from synthesis of an amyloid precursor protein to its conversion to the amyloid fiber subunit. We address this question by characterizing the effects of an amyloidogenic mutation on the intracellular processing of its protein product. The protein, a mutant of the cysteine protease inhibitor cystatin C, is the amyloid precursor protein in Hereditary Cerebral Hemorrhage with Amyloidosis--Icelandic type (HCHWA-I). The amyloid fibers are composed of mutant cystatin C (L68Q) that lacks the first 10 amino acids. We have previously shown that processing of wild-type cystatin C entails formation of a transient intracellular dimer that dissociates prior to secretion, such that extracellular cystatin C is monomeric. We report here that the cystatin C mutation engenders several alterations in its intracellular trafficking. It forms a stable intracellular dimer that is partially retained in the endoplasmic reticulum and degraded. The bulk of mutant cystatin C that is secreted does not dissociate and is secreted as an inactive dimer. Thus, formation of the stable mutant cystatin C dimer is an early event in the pathogenesis of this disease.

Tau proteins are central to the neuropathology of Alzheimer’s disease and tau levels in cerebrospinal fluid are elevated in affected individuals. In this study, we investigated the presence of tau in plasma from subjects with Alzheimer’s disease (n = 16), frontotemporal dementia (n = 10), vascular dementia (n = 16) and from healthy controls (n = 15). By using an ELISA with monoclonal tau antibodies, tau immunoreactivity was detected in approximately 20% of the subjects. However, no difference between the disease and control groups was seen. After gel filtration of tau immunopositive plasma, the peak reactivity was found in the 160-kD fraction, indicating the source to be tau-like molecules of high-molecular-weight or polymers of low-molecular-weight tau isoforms. We conclude that measurements of tau in plasma cannot be utilized diagnostically for Alzheimer’s disease or for the other dementias investigated.

To define the cellular processing of human cystatin C as well as to lay the groundwork for investigating its contribution to lcelandic Hereditary Cerebral Hemorrhage with Amyloidosis (HCHWA-I), we have characterized the trafficking, secretion, and extracellular fate of human cystatin C in transfected Chinese hamster ovary (CHO) cells. It is constitutively secreted with an intracellular half-life of 72 min. Gel filtration of cell lysates revealed the presence of three cystatin C immunoreactive species; an 11 kDa species corresponding to monomeric cystatin C, a 33 kDa complex that is most likely dimeric cystatin C and immunoreactive material, > or = 70 kDa, whose composition is unknown. Intracellular monomeric cystatin C is functionally active as a cysteine protease inhibitor, while the dimer is not. Medium from the transfected CHO cells contained only active monomeric cystatin C indicating that the cystatin C dimer, formed during intracellular trafficking, is converted to monomer at or before secretion. Cells in which exit from the endoplasmic reticulum (ER) was blocked with brefeldin A contained the 33 kDa species, indicating that cystatin C dimerization occurs in the ER. After removal of brefeldin A, there was a large increase in intracellular monomer suggesting that dimer dissociation occurs later in the secretion pathway, after exiting the ER but prior to release from the cell. Extracellular monomeric cystatin C was found to be internalized into lysosomes where it again dimerized, presumably as a consequence of the low pH of late endosome/lysosomes. As a dimer, cystatin C would be prevented from inhibiting the lysosomal cysteine proteases. These results reveal a novel mechanism, transient dimerization, by which cystatin C is inactivated during the early part of its trafficking through the secretory pathway and then reactivated prior to secretion. Similarly, its uptake by the cell also leads to its redimerization in the lysosomal pathway.

IntroductionThe diagnoses of autism spectrum disorders (ASDs) are based on a phenotype, characterized by impaired social interaction and communication and by repetitive and restricted interests. However, this might not represent a single clinical entity, but a behavioral manifestation of different neurodevelopmental deficits with a multifactorial etiology. Small studies have shown elevated levels of oxidative stress and lower levels of anti-oxidants in patients with ASD, and correlations with the severity of ASD. Therapies targeting oxidative stress have shown improvements regarding behavior, social interaction and verbal communication in patients with ASD, supporting the oxidative stress theory.ObjectivesTo evaluate the importance of oxidative stress in the neurobiology of adults with ASD.AimsThere is a need to understand the neurobiology of ASD, therefore this study analyzes the level of oxidative stress in a larger cohort of patients with ASD and compares to controls.MethodsThe study includes 350 patients over 18 years of age diagnosed with ICD-10 diagnoses F84.0, F84.1, F84.5 or F84.8 and compared to gender and age matched neurotypical controls. The included probands will have their serum and plasma analyzed for levels of oxidative stress (superoxide dismutase 1 and 2, catalase, glutathioneperoxidase, malonialdehyde, thiobarbituric acid reactive substances and xanthinoxidase).ResultsThe preliminary results will be presented at the EPA in March 2016 in Madrid.ConclusionWith this study we aim to elucidate some of the neurobiology in ASD. This could lead to new potential targets for treatment and prevention of the disorders.Disclosure of interestThe authors have not supplied their declaration of competing interest.

The proteasome is important for degradation of worn out and misfolded proteins. Decreased proteasome activity has been implicated in Alzheimer's disease (AD). Proteasome inhibition induces autophagy, but it is still unknown whether autophagy is beneficial or deleterious to AD neurons, as the autophagosome has been suggested as a site of amyloid-β (Aβ) generation. In this study, we investigated the effect of proteasome inhibition on Aβ accumulation and secretion, as well as the processing of amyloid-β protein precursor (AβPP) in AβPP(Swe) transfected SH-SY5Y neuroblastoma cells. We show that proteasome inhibition resulted in autophagy-dependent accumulation of Aβ in lysosomes, and increased levels of intracellular and secreted Aβ. The enhanced levels of Aβ could not be explained by increased amounts of AβPP. Instead, reduced degradation of the C-terminal fragment of AβPP (C99) by the proteasome makes C99 available for γ-secretase cleavage, leading to Aβ generation. Inhibition of autophagy after proteasome inhibition led to reduced levels of intracellular, but not secreted Aβ, and tended to further increase the C99 to AβPP ratio, supporting involvement of the autophagosome in Aβ generation. Furthermore, proteasome inhibition caused a reduction in cellular viability, which was reverted by inhibition of autophagy. Dysfunction of the proteasome could cause lysosomal accumulation of Aβ, as well as increased generation and secretion of Aβ, which is partly facilitated by autophagy. As a decrease in cellular viability was also detected, it is possible that upregulation of autophagy is an unsuccessful rescue mechanism, which instead of being protective, contributes to AD pathogenesis.

A total of 66 skin biopsies from persons with Alzheimer's disease (AD) or Down's syndrome (DS) and from persons without AD were used in this study. The age range was from 7 to 89 years. Positive immunoreactivity of skin biopsies to monoclonal antibody 4G8, which is reactive to amino acid residue 17-24 of synthetic amyloid beta protein (A beta), and 4G8-Fab (the antigen-binding fragment of 4G8 IgG, reactive only to amyloid plaque) was observed in the epidermis-dermis junction or the basement membrane of the epidermis and in some blood vessels of the biopsy skins of 13/18 (72%) AD, 9/10 (90%) DS, and 14/38 (37%) non-AD control cases. The Fisher exact probability test revealed a significant difference (P = 0.0415 one-tailed) in immunoreactivity between AD and age-matched controls. There was also a significant difference (P = 0.0152 one-tailed; P = 0.0200 two-tailed) between DS and age-matched control in the same test. Immuno-gold electron microscopy examination of these cases with positive immunoreactivity revealed that the gold particles were deposited along the basement membrane of the epidermis. Amyloid fibrils were not observed in the regions with gold particles. Results of this study suggest that A beta is associated with the basement membrane of skin and is present in amorphous, non-fibrillar form as soluble A beta.

Increasing evidence suggests the toxicity of intracellular amyloid β-protein (Aβ) to neurons, as well as the involvement of oxidative stress in Alzheimer disease (AD). Here we show that normobaric hyperoxia (exposure of cells to 40% oxygen for five days, and consequent activation of macroautophagy and accumulation of Aβ within lysosomes, induced apoptosis in differentiated SH-SY5Y neuroblastoma cells. Cells under hyperoxia showed: (1) increased numbers of autophagic vacuoles that contained amyloid precursor protein (APP) as well as Aβ monomers and oligomers, (2) increased reactive oxygen species production, and (3) enhanced apoptosis. Oxidant-induced apoptosis positively correlated with cellular Aβ production, being the highest in cells that were stably transfected with APP Swedish KM670/671NL double mutation. Inhibition of γ-secretase, prior and/or in parallel to hyperoxia, suggested that the increase of lysosomal Aβ resulted mainly from its autophagic uptake, but also from APP processing within autophagic vacuoles. The oxidative stress-mediated effects were prevented by macroautophagy inhibition using 3-methyladenine or ATG5 downregulation. Our results suggest that upregulation of macroautophagy and resulting lysosomal Aβ accumulation are essential for oxidant-induced apoptosis in cultured neuroblastoma cells and provide aditional support for the interactive role of oxidative stress and the lysosomal system in AD-related neurodegeneration. Funding agencies|Gustav V and Queen Victoria Foundation||County Council of Ostergotland||Stiftelsen Olle Engkvist Byggmastare||Stifielsen for Gamla Tjanarinnor||Gunoch Bertil Stohnes Stiftelse||Lions forskningsfond||Svenska Lundbeckstiftelsen||Karolinska Institute Fund for Geriatric Research||Alice och Knut Wallenberg Stiftelse||Swedish Alzheimer Foundation||Swedish Brain Power

Recently, interest in the rat as an animal model of Alzheimer’s disease (AD) has been growing. We have previously described the Tg6590 transgenic rat line expressing the amyloid precursor protein containing the Swedish AD mutation (K670M/N671L) that shows early stages of Aβ deposition, predominantly in cerebrovascular blood vessels, after 15 months of age. Here we show that by the age of 9 months, that is long before the appearance of Aβ deposits, the Tg6590 rats exhibit deficits in the Morris water maze spatial navigation task and altered spontaneous behaviour in the open-field test. The levels of soluble Aβ were elevated both in the hippocampus and cortex of transgenic animals. Magnetic resonance imaging showed no major changes in the brains of transgenic animals, although they tended to have enlarged lateral ventricles when compared to control animals. The Tg6590 transgenic rat line should prove a suitable model of early AD for advanced studies including serial cerebrospinal fluid sampling, electrophysiology, neuroimaging or complex behavioural testing.

There is increasing evidence for the toxicity of intracellular amyloid beta-protein (Abeta) to neurons and the involvement of lysosomes in this process in Alzheimer disease (AD). We have recently shown that oxidative stress, a recognized determinant of AD, enhances macroautophagy and leads to intralysosomal accumulation of Abeta in cultured neuroblastoma cells. We hypothesized that oxidative stress promotes AD by stimulating macroautophagy of Abeta that further may induce cell death by destabilizing lysosomal membranes. To investigate such possibility, we compared the effects of hyperoxia (40% ambient oxygen) in cultured HEK293 cells that were transfected with an empty vector (Vector), wild-type APP (APPwt), or Swedish mutant APP (APPswe). Exposure to hyperoxia for 5 days increased the number of cells with Abeta-containing lysosomes, as well as the number of apoptotic cells, compared to normoxic conditions. The rate of apoptosis in all three cell lines demonstrated dependence on intralysosomal Abeta content (Vector

Altered calcium homeostasis is implicated in the pathogenesis of Alzheimer's disease and much effort has been put into understanding the association between the autosomal dominant gene mutations causative of this devastating disease and perturbed calcium signaling. We have focused our attention on the effect of the APP670/671 mutation on spontaneous calcium oscillations in embryonic hippocampal neurons derived from the tg6590 transgenic rat. Intracellular free calcium levels were imaged by confocal microscopy using the fluorescent dye fluo-3AM. Hyperosmotic shrinkage, which can occur in a variety of pathophysiological conditions, has been shown to induce multiple cellular responses, including activation of volume-regulatory ion transport, cytoskeletal reorganization, and cell death. When exposed to hyperosmotic stress (addition of 50 mM sucrose) the frequency of calcium oscillations was suppressed to an equal extent in both wild-type and transgenic cultures, but the transgenic neurons, in contrast to the wild-type neurons, responded with a significantly higher increase in the amplitude of oscillations. A decrease in cell viability was observed by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay in neurons exposed to the hyperosmotic medium for 30 h. Whereas this loss in cell viability was comparable in both sets of cultures, the amplitude of calcium oscillations in transgenic neurons exhibited a significantly greater decrease in the presence of the L-type calcium channel antagonist, nimodipine. These results suggest that APP670/671 transgenic neurons have impaired calcium homeostasis.

When screening a brain cDNA library, we found that the N-methyl-D-aspartate receptor subunit NR3A binds to microtubule-associated protein (MAP) 1S/chromosome 19 open reading frame 5 (C19ORF5). The interaction was confirmed in vitro and in vivo, and binding of MAP1S was localized to the membrane-proximal part of the NR3A C-terminus. MAP1S belongs to the same family as MAP1A and MAP1B, and was found to be abundant in both postnatal and adult rat brain. In hippocampal neurons the distribution-pattern of MAP1S resembled that of beta-tubulin III, but a fraction of the protein colocalized with synaptic markers synapsin and postsynaptic density protein 95 (PSD95), in beta-tubulin III-negative filopodia-like protrusions. There was coexistance between MAP1S and NR3A immunoreactivity in neurite shafts and occasionally in filopodia-like processes. MAP1S potentially links NR3A to the cytoskeleton, and may stabilize NR3A-containing receptors at the synapse and regulate their movement between synaptic and extrasynaptic sites.

Presenilins (PSs) are involved in processing several proteins such as the amyloid precursor protein (APP), as well as in pathways for cell death and survival. We previously showed that some familial Alzheimer's disease PS mutations cause increased basal and acetylcholine muscarinic receptor-stimulated phospholipase C (PLC) activity which was gamma-secretase dependent. To further evaluate the dependence of PLC on PSs we measured PLC activity and the activation of variant protein kinase C (PKC) isoforms in mouse embryonic fibroblasts (MEFs) lacking either PS1, PS2, or both. PLC activity and PKCalpha and PKCgamma activations were significantly lower in PS1 and PS2 double knockout MEFs after PLC stimulation. Protein levels of PKCalpha and PKCgamma were lower in PS1 and PS2 double knockout MEFs. In contrast, PKCdelta levels were significantly elevated in PS1 and PS2 double knockout as well as in PS1 knockout MEFs. Also, PKCdelta levels were lowered after transfection of PS1 into PS1 knockout or PS double knockout MEFs. Using APP knockout MEFs we showed that the expression of PKCalpha, but not the other PKC isoforms is partially dependent on APP and can be regulated by APP intracellular domain (AICD). These results show that PLC and PKC activations are modulated by PS and also that PSs differentially regulate the expression of PKC isoforms by both APP/AICD-dependent and independent mechanisms.

In recent years, transgenic mice have become valuable tools for studying mechanisms of Alzheimer's disease (AD). With the aim of developing an animal model better for memory and neurobehavioural testing, we have generated a transgenic rat model of AD. These animals express human amyloid precursor protein (APP) containing the Swedish AD mutation. The highest level of expression in the brain is found in the cortex, hippocampus, and cerebellum. Starting after the age of 15 months, the rats show increased tau phosphorylation and extracellular Abeta staining. The Abeta is found predominantly in cerebrovascular blood vessels with very rare diffuse plaques. We believe that crossing these animals with mutant PS1 transgenic rats will result in accelerated plaque formation similar to that seen in transgenic mice.

A single nucleotide polymorphism in the ZNF804A gene, rs1344706, is associated with schizophrenia. The polymorphism has been suggested to alter fetal expression of ZNF804A. It has also been reported to be associated with altered cortical functioning and neural connectivity in the brain. Since developmental mechanisms are suggested in the pathophysiology for schizophrenia, expression of Zfp804A, the rat homolog of ZNF804A, was investigated in the developing rat brain. We found that expression of Zfp804A in most brain regions is developmentally regulated and peaks around birth, where after it decreases towards adult levels. This time point is developmentally the equivalent to the second trimester of fetal development in humans. An exception to this expression pattern is the hippocampus where the expression of Zfp804A appears to increase again in the adult brain. Using laser capture and quantitative PCR we found that Zfp804A mRNA expression in the adult rat hippocampus is highest in the CA1 sub region, where the overall firing rates of neurons is higher than in the CA3 region. In cultured cortical neurons Zfp804A mRNA expression peaked at day 4 and then decreased. The ZFP804A protein expression was therefore investigated with immunochemistry in such cultures. Interestingly, before day 4, the protein is mostly found in the perinuclear region of the cell but at day 4, ZFP804A was instead found throughout the cell and particularly in the growth cones. In conclusion we demonstrate that Zfp804A increases in the rat brain at the time of birth, coinciding with neuronal differentiation. We also show that ZFP804A is localized to growth cones of growing neurites. These data implicate ZFP804A in growth cone function and neurite elongation. The polymorphism rs1344706 lowers expression of ZNF804A during prenatal brain development. This may affect ZNF804A's role in cone function and neurite elongation leading to synaptic deficits and altered neural connectivity.

In this study, we characterise the binding site of the human N -methyl- d -aspartate (NMDA) receptor subunit NR3A. Saturation radioligand binding of the NMDA receptor agonists [ 3 H]-glycine and [ 3 H]-glutamate showed that only glycine binds to human NR3A (hNR3A) with high affinity ( K d = 535 nM (277–793 nM)). Eight amino acids, which correspond to amino acids that are critical for ligand binding to other NMDA receptor subunits, situated within the S1S2 predicted ligand binding domain of hNR3A were mutated, which resulted in complete or near complete loss of [ 3 H]-glycine binding to hNR3A. The NMDA NR1 glycine site agonist d -serine and partial agonist HA-966 (3-amino-1-hydroxypyrrolid-2-one), similarly to glycine displaced [ 3 H]-glycine monophasically, suggesting a single common binding site. However, neither the partial agonist d -cycloserine nor the antagonist 7-chlorokynurenic acid displaced [ 3 H]-glycine. Using homology modelling, a model of the NR3A binding pocket was generated which we suggest can be used to identify candidate agonists and antagonists. Our data show that glycine is a ligand, and most probably the endogenous ligand, for native NR3A at a binding site with unique pharmacological characteristics.

The amyloid precursor protein can through ligand-mimicking induce expression of ornithine decarboxylase (ODC), the initial and rate-limiting enzyme in polyamine biosynthesis. We report here the regional distribution and cellular localization of ODC immunoreactivity in Alzheimer's disease (AD) brains. In frontal cortex and hippocampus of control cases, the most pronounced ODC immunoreactivity was found in the nucleus. In possible and definite AD the immunoreactivity had shifted to the cytoplasm. In cerebellum of control cases, ODC staining was found in a small portion of Purkinje cells, mostly in the nucleus. In AD, both possible and definite, the number of stained Purkinje cells increased significantly and immunoreactivity was shifted to the cytoplasm, even though it was still prominent in the nucleus. In conclusion, our study reveals an early shift of the ODC immunoreactivity in AD from the nuclear compartment towards the cytoplasm.

Polychlorinated Biphenyls (PCBs)-induced changes in synaptic transmission are one of the effects of their neurotoxicity but the mechanism remains unknown. We assessed the in vivo effects of the PCBs mixture, Aroclor 1254 on the expression of neuronal proteins that are involved in the synaptic function and/or are associated with neurodegeneration. Wistar rats were treated orally with repeated doses of Aroclor 1254 and the levels of soluble alpha-synuclein, parkin, synaptophysin and amyloid precursor protein (APP) in the brain were determined by Western blotting. The results showed that Aroclor did not cause changes in the expression and processing of APP but at a dose 100 microg/g/day repeated for 6 days caused a decrease in the expression of alpha-synuclein in the cerebellum, cortex, hippocampus and hypothalamus of the animals sacrificed 2 days after treatment. The decrease in alpha-synuclein was accompanied by a transient increase in parkin and synaptophysin levels. Interestingly, in the hypothalamus the levels of alpha-synuclein remained decreased after 21 days post treatment perhaps due to regional differences in the PCBs elimination or perhaps a more specific interaction with the dopaminergic cells that are present in the hypothalamus that needs to be investigated further.