BACE inhibitor treatment of mice induces hyperactivity in a Seizure-related gene 6 family dependent manner without altering learning and memory

BACE inhibitors, which decrease BACE1 (beta -secretase 1) cleavage of the amyloid precursor protein, are a potential treatment for Alzheimer's disease. Clinical trials using BACE inhibitors have reported a lack of positive effect on patient symptoms and, in some cases, have led to increased adverse events, cognitive worsening and hippocampal atrophy. A potential drawback of this strategy is the effect of BACE inhibition on other BACE1 substrates such as Seizure-related gene 6 (Sez6) family proteins which are known to have a role in neuronal function. Mice were treated with an in-diet BACE inhibitor for 4-8 weeks to achieve a clinically-relevant level of amyloid-beta 40 reduction in the brain. Mice underwent behavioural testing and postmortem analysis of dendritic spine number and morphology with Golgi-Cox staining. Sez6 family triple knockout mice were tested alongside wild-type mice to identify whether any effects of the treatment were due to altered cleavage of Sez6 family proteins. Wild-type mice treated with BACE inhibitor displayed hyperactivity on the elevated open field, as indicated by greater distance travelled, but this effect was not observed in treated Sez6 triple knockout mice. BACE inhibitor treatment did not lead to significant changes in spatial or fear learning, reference memory, cognitive flexibility or anxiety in mice as assessed by the Morris water maze, context fear conditioning, or light-dark box tests. Chronic BACE inhibitor treatment reduced the density of mushroom-type spines in the somatosensory cortex, regardless of genotype, but did not affect steady-state dendritic spine density or morphology in the CA1 region of the hippocampus. Chronic BACE inhibition for 1-2 months in mice led to increased locomotor output but did not alter memory or cognitive flexibility. While the mechanism underlying the treatment-induced hyperactivity is unknown, the absence of this response in Sez6 triple knockout mice indicates that blocking ectodomain shedding of Sez6 family proteins is a contributing factor. In contrast, the decrease in mature spine density in cortical neurons was not attributable to lack of shed Sez6 family protein ectodomains. Therefore, other BACE1 substrates are implicated in this effect and, potentially, in the cognitive decline in longer-term chronically treated patients

The Speciality Index as invariant indicator in the BKL Mixmaster Dynamics

The speciality index, which has been mainly used in Numerical Relativity for studying gravitational waves phenomena as an indicator of the special or non-special Petrov type character of a spacetime, is applied here in the context of Mixmaster cosmology, using the Belinski-Khalatnikov-Lifshitz map. Possible applications for the associated chaotic dynamics are discussed

The beta-Secretase Substrate Seizure 6-Like Protein (SEZ6L) Controls Motor Functions in Mice

The membrane protein seizure 6–like (SEZ6L) is a neuronal substrate of the Alzheimer’s disease protease BACE1, and little is known about its physiological function in the nervous system. Here, we show that SEZ6L constitutive knockout mice display motor phenotypes in adulthood, including changes in gait and decreased motor coordination. Additionally, SEZ6L knockout mice displayed increased anxiety-like behaviour, although spatial learning and memory in the Morris water maze were normal. Analysis of the gross anatomy and proteome of the adult SEZ6L knockout cerebellum did not reveal any major differences compared to wild type, indicating that lack of SEZ6L in other regions of the nervous system may contribute to the phenotypes observed. In summary, our study establishes physiological functions for SEZ6L in regulating motor coordination and curbing anxiety-related behaviour, indicating that aberrant SEZ6L function in the human nervous system may contribute to movement disorders and neuropsychiatric diseases

MicroSAGE is Highly Representative and Reproducible but Reveals Major Differences in Gene Expression Among Samples Obtained from Similar Tissues

Background: Serial analysis of gene expression using small amounts of starting material (microSAGE) has not yet been conclusively shown to be representative, reproducible or accurate. Results: We show that microSAGE is highly representative, reproducible and accurate, but that pronounced differences in gene expression are seen between tissue samples taken from different individuals. Conclusions: MicroSAGE is a reliable method of comprehensively profiling differences in gene expression among samples, but care should be taken in generalizing results obtained from libraries constructed from tissue obtained from different individuals and/or processed or stored differently

Seizure protein 6 and its homolog seizure 6-like protein are physiological substrates of BACE1 in neurons

Background: The protease BACE1 (beta-site APP cleaving enzyme) is a major drug target in Alzheimer’s disease. However, BACE1 therapeutic inhibition may cause unwanted adverse effects due to its additional functions in the nervous system, such as in myelination and neuronal connectivity. Additionally, recent proteomic studies investigating BACE1 inhibition in cell lines and cultured murine neurons identified a wider range of neuronal membrane proteins as potential BACE1 substrates, including seizure protein 6 (SEZ6) and its homolog SEZ6L. Methods and results: We generated antibodies against SEZ6 and SEZ6L and validated these proteins as BACE1 substrates in vitro and in vivo. Levels of the soluble, BACE1-cleaved ectodomain of both proteins (sSEZ6, sSEZ6L) were strongly reduced upon BACE1 inhibition in primary neurons and also in vivo in brains of BACE1-deficient mice. BACE1 inhibition increased neuronal surface levels of SEZ6 and SEZ6L as shown by cell surface biotinylation, demonstrating that BACE1 controls surface expression of both proteins. Moreover, mass spectrometric analysis revealed that the BACE1 cleavage site in SEZ6 is located in close proximity to the membrane, similar to the corresponding cleavage site in SEZ6L. Finally, an improved method was developed for the proteomic analysis of murine cerebrospinal fluid (CSF) and was applied to CSF from BACE-deficient mice. Hereby, SEZ6 and SEZ6L were validated as BACE1 substrates in vivo by strongly reduced levels in the CSF of BACE1-deficient mice. Conclusions: This study demonstrates that SEZ6 and SEZ6L are physiological BACE1 substrates in the murine brain and suggests that sSEZ6 and sSEZ6L levels in CSF are suitable markers to monitor BACE1 inhibition in mice

Seizure protein 6 and its homolog seizure 6-like protein are physiological substrates of BACE1 in neurons

Background: The protease BACE1 (beta-site APP cleaving enzyme) is a major drug target in Alzheimer’s disease. However, BACE1 therapeutic inhibition may cause unwanted adverse effects due to its additional functions in the nervous system, such as in myelination and neuronal connectivity. Additionally, recent proteomic studies investigating BACE1 inhibition in cell lines and cultured murine neurons identified a wider range of neuronal membrane proteins as potential BACE1 substrates, including seizure protein 6 (SEZ6) and its homolog SEZ6L. Methods and results: We generated antibodies against SEZ6 and SEZ6L and validated these proteins as BACE1 substrates in vitro and in vivo. Levels of the soluble, BACE1-cleaved ectodomain of both proteins (sSEZ6, sSEZ6L) were strongly reduced upon BACE1 inhibition in primary neurons and also in vivo in brains of BACE1-deficient mice. BACE1 inhibition increased neuronal surface levels of SEZ6 and SEZ6L as shown by cell surface biotinylation, demonstrating that BACE1 controls surface expression of both proteins. Moreover, mass spectrometric analysis revealed that the BACE1 cleavage site in SEZ6 is located in close proximity to the membrane, similar to the corresponding cleavage site in SEZ6L. Finally, an improved method was developed for the proteomic analysis of murine cerebrospinal fluid (CSF) and was applied to CSF from BACE-deficient mice. Hereby, SEZ6 and SEZ6L were validated as BACE1 substrates in vivo by strongly reduced levels in the CSF of BACE1-deficient mice. Conclusions: This study demonstrates that SEZ6 and SEZ6L are physiological BACE1 substrates in the murine brain and suggests that sSEZ6 and sSEZ6L levels in CSF are suitable markers to monitor BACE1 inhibition in mice

Seizure-Related Gene 6 (Sez-6) in Amacrine Cells of the Rodent Retina and the Consequence of Gene Deletion

Background: Seizure-related gene 6 (Sez-6) is expressed in neurons of the mouse brain, retina and spinal cord. In the cortex, Sez-6 plays a role in specifying dendritic branching patterns and excitatory synapse numbers during development. Methodology/Principal Findings: The distribution pattern of Sez-6 in the retina was studied using a polyclonal antibody that detects the multiple isoforms of Sez-6. Prominent immunostaining was detected in GABAergic, but not in All glycinergic, amacrine cell subpopulations of the rat and mouse retina. Amacrine cell somata displayed a distinct staining pattern with the Sez-6 antibody: a discrete, often roughly triangular-shaped bright spot positioned between the nucleus and the apical dendrite superimposed over weaker general cytoplasmic staining. Displaced amacrines in the ganglion cell layer were also positive for Sez-6 and weaker staining was occasionally observed in neurons with the morphology of alpha ganglion cells. Two distinct Sez-6 positive strata were present in the inner plexiform layer in addition to generalized punctate staining. Certain inner nuclear layer cells, including bipolar cells, stained more weakly and diffusely than amacrine cells, although some bipolar cells exhibited a perinuclear "bright spot" similar to amacrine cells. In order to assess the role of Sez-6 in the retina, we analyzed the morphology of the Sez-6 knockout mouse retina with immunohistochemical markers and compared ganglion cell dendritic arbor patterning in Sez-6 null retinae with controls. The functional importance of Sez-6 was assessed by dark-adapted paired-flash electroretinography (ERG). Conclusions: In summary, we have reported the detailed expression pattern of a novel retinal marker with broad cell specificity, useful for retinal characterization in rodent experimental models. Retinal morphology, ganglion cell dendritic branching and ERG waveforms appeared normal in the Sez-6 knockout mouse suggesting that, in spite of widespread expression of Sez-6, retinal function in the absence of Sez-6 is not affected

Seizure protein 6 controls glycosylation and trafficking of kainate receptor subunits GluK2 and GluK3

Seizure protein 6 (SEZ6) is required for the development and maintenance of the nervous system, is a major substrate of the protease BACE1 and is linked to Alzheimer's disease (AD) and psychiatric disorders, but its molecular functions are not well understood. Here, we demonstrate that SEZ6 controls glycosylation and cell surface localization of kainate receptors composed of GluK2/3 subunits. Loss of SEZ6 reduced surface levels of GluK2/3 in primary neurons and reduced kainate-evoked currents in CA1 pyramidal neurons in acute hippocampal slices. Mechanistically, loss of SEZ6 in vitro and in vivo prevented modification of GluK2/3 with the human natural killer-1 (HNK-1) glycan, a modulator of GluK2/3 function. SEZ6 interacted with GluK2 through its ectodomain and promoted post-endoplasmic reticulum transport of GluK2 in the secretory pathway in heterologous cells and primary neurons. Taken together, SEZ6 acts as a new trafficking factor for GluK2/3. This novel function may help to better understand the role of SEZ6 in neurologic and psychiatric diseases