Transgenic neuronal overexpression reveals that stringently regulated p23 expression is critical for coordinated movement in mice

<p>Abstract</p> <p>Background</p> <p>p23 belongs to the highly conserved p24 family of type I transmembrane proteins, which participate in the bidirectional protein transport between the endoplasmic reticulum and Golgi apparatus. Mammalian p23 has been shown to interact with γ-secretase complex, and modulate secretory trafficking as well as intramembranous processing of amyloid precursor protein in cultured cells. Negative modulation of β-amyloid production by p23 in cultured cell lines suggested that elevation of p23 expression in neurons might mitigate cerebral amyloid burden.</p> <p>Results</p> <p>We generated several lines of transgenic mice expressing human p23 in neurons under the control of <it>Thy-1.2 </it>promoter. We found that even a 50% increase in p23 levels in the central nervous system of mice causes post-natal growth retardation, severe neurological problems characterized by tremors, seizure, ataxia, and uncoordinated movements, and premature death. The severity of the phenotype closely correlated with the level of p23 overexpression in multiple transgenic lines. While the number and general morphology of neurons in Hup23 mice appeared to be normal throughout the brain, abnormal non-Golgi p23 localization was observed in a subset of neurons with high transgene expression in brainstem. Moreover, detailed immunofluorescence analysis revealed marked proliferation of astrocytes, activation of microglia, and thinning of myelinated bundles in brainstem of Hup23 mice.</p> <p>Conclusions</p> <p>These results demonstrate that proper level of p23 expression is critical for neuronal function, and perturbing p23 function by overexpression initiates a cascade of cellular reactions in brainstem that leads to severe motor deficits and other neurological problems, which culminate in premature death. The neurological phenotype observed in Hup23 mice highlights significant adverse effects associated with manipulating neuronal expression of p23, a previously described negative modulator of γ-secretase activity and β-amyloid production. Moreover, our report has broader relevance to molecular mechanisms in several neurodegenerative diseases as it highlights the inherent vulnerability of the early secretory pathway mechanisms that ensure proteostasis in neurons.</p

Axonal BACE1 dynamics and targeting in hippocampal neurons: a role for Rab11 GTPase

BACKGROUND: BACE1 is one of the two enzymes that cleave amyloid precursor protein to generate Alzheimer's disease (AD) beta amyloid peptides. It is widely believed that BACE1 initiates APP processing in endosomes, and in the brain this cleavage is known to occur during axonal transport of APP. In addition, BACE1 accumulates in dystrophic neurites surrounding brain senile plaques in individuals with AD, suggesting that abnormal accumulation of BACE1 at presynaptic terminals contributes to pathogenesis in AD. However, only limited information is available on BACE1 axonal transport and targeting. RESULTS: By visualizing BACE1-YFP dynamics using live imaging, we demonstrate that BACE1 undergoes bi-directional transport in dynamic tubulo-vesicular carriers along axons in cultured hippocampal neurons and in acute hippocampal slices of transgenic mice. In addition, a subset of BACE1 is present in larger stationary structures, which are active presynaptic sites. In cultured neurons, BACE1-YFP is preferentially targeted to axons over time, consistent with predominant in vivo localization of BACE1 in presynaptic terminals. Confocal analysis and dual-color live imaging revealed a localization and dynamic transport of BACE1 along dendrites and axons in Rab11-positive recycling endosomes. Impairment of Rab11 function leads to a diminution of total and endocytosed BACE1 in axons, concomitant with an increase in the soma. Together, these results suggest that BACE1 is sorted to axons in endosomes in a Rab11-dependent manner. CONCLUSION: Our results reveal novel information on dynamic BACE1 transport in neurons, and demonstrate that Rab11-GTPase function is critical for axonal sorting of BACE1. Thus, we suggest that BACE1 transcytosis in endosomes contributes to presynaptic BACE1 localization

Dual roles of the transmembrane protein p23/TMP21 in the modulation of amyloid precursor protein metabolism

BACKGROUND: Alzheimer's disease (AD) is characterized by cerebral deposition of β-amyloid (Aβ) peptides. Aβ is released from ectodomain cleaved amyloid precursor protein (APP) via intramembranous proteolysis by γ-secretase, a complex consisting of presenilin and a few other proteins. p23/TMP21, a member of the p24 family type I transmembrane proteins, was recently identified as a presenilin complex component capable of modulating γ-secretase cleavage. The p24 family proteins form oligomeric complexes and regulate vesicular trafficking in the early secretory pathway, but their role in APP trafficking has not been investigated. RESULTS: Here, we report that siRNA-mediated depletion of p23 in N2a neuroblastoma and HeLa cells produces concomitant knockdown of additional p24 family proteins and increases secretion of sAPP. Furthermore, intact cell and cell-free Aβ production increases following p23 knockdown, similar to data reported earlier using HEK293 cells. However, we find that p23 is not present in mature γ-secretase complexes isolated using an active-site γ-secretase inhibitor. Depletion of p23 and expression of a familial AD-linked PS1 mutant have additive effects on Aβ(42 )production. Knockdown of p23 expression confers biosynthetic stability to nascent APP, allowing its efficient maturation and surface accumulation. Moreover, immunoisolation analyses show decrease in co-residence of APP and the APP adaptor Mint3. Thus, multiple lines of evidence indicate that p23 function influences APP trafficking and sAPP release independent of its reported role in γ-secretase modulation. CONCLUSION: These data assign significance to p24 family proteins in regulating APP trafficking in the continuum of bidirectional transport between the ER and Golgi, and ascribe new relevance to the regulation of early trafficking in AD pathogenesis

Loss of Cleavage at β′-Site Contributes to Apparent Increase in β-Amyloid Peptide (Aβ) Secretion by β-Secretase (BACE1)-Glycosylphosphatidylinositol (GPI) Processing of Amyloid Precursor Protein

Several lines of evidence implicate lipid raft microdomains in Alzheimer disease-associated β-amyloid peptide (Aβ) production. Notably, targeting β-secretase (β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1)) exclusively to lipid rafts by the addition of a glycosylphosphatidylinositol (GPI) anchor to its ectodomain has been reported to elevate Aβ secretion. Paradoxically, Aβ secretion is not reduced by the expression of non-raft resident S -palmitoylation-deficient BACE1 (BACE1-4C/A (C474A/C478A/C482A/C485A)). We addressed this apparent discrepancy in raft microdomain-associated BACE1 processing of APP in this study. As previously reported, we found that expression of BACE1-GPI elevated Aβ secretion as compared with wild-type BACE1 (WTBACE1) or BACE1-4C/A. However, this increase occurred without any difference in the levels of APP ectodomain released following BACE1 cleavage (soluble APPβ), arguing against an overall increase in BACE1 processing of APP per se . Further analysis revealed that WTBACE1 cleaves APP at β- and β′-sites, generating +1 and +11 β-C-terminal fragments and secreting intact as well as N-terminally truncated Aβ. In contrast, three different BACE1-GPI chimeras preferentially cleaved APP at the β-site, mainly generating +1 β-C-terminal fragment and secreting intact Aβ. As a consequence, cells expressing BACE1-GPI secreted relatively higher levels of intact Aβ without an increase in BACE1 processing of APP. Markedly reduced cleavage at β′-site exhibited by BACE1-GPI was cell type-independent and insensitive to subcellular localization of APP or the pathogenic KM/NL mutant. We conclude that the apparent elevation in Aβ secretion by BACE1-GPI is mainly attributed to preferential cleavage at the β-site and failure to detect +11 Aβ species secreted by cells expressing WTBACE1

Ca2+ influx through store-operated Ca2+ channels reduces Alzheimer disease β-amyloid peptide secretion.

Alzheimer disease (AD), the leading cause of dementia, is characterized by the accumulation of β-amyloid peptides (Aβ) in senile plaques in the brains of affected patients. Many cellular mechanisms are thought to play important roles in the development and progression of AD. Several lines of evidence point to the dysregulation of Ca(2+) homeostasis as underlying aspects of AD pathogenesis. Moreover, direct roles in the regulation of Ca(2+) homeostasis have been demonstrated for proteins encoded by familial AD-linked genes such as PSEN1, PSEN2, and APP, as well as Aβ peptides. Whereas these studies support the hypothesis that disruption of Ca(2+) homeostasis contributes to AD, it is difficult to disentangle the effects of familial AD-linked genes on Aβ production from their effects on Ca(2+) homeostasis. Here, we developed a system in which cellular Ca(2+) homeostasis could be directly manipulated to study the effects on amyloid precursor protein metabolism and Aβ production. We overexpressed stromal interaction molecule 1 (STIM1) and Orai1, the components of the store-operated Ca(2+) entry pathway, to generate cells with constitutive and store depletion-induced Ca(2+) entry. We found striking effects of Ca(2+) entry induced by overexpression of the constitutively active STIM1(D76A) mutant on amyloid precursor protein metabolism. Specifically, constitutive activation of Ca(2+) entry by expression of STIM1(D76A) significantly reduced Aβ secretion. Our results suggest that disruptions in Ca(2+) homeostasis may influence AD pathogenesis directly through the modulation of Aβ production

Ca2+ influx through store-operated Ca2+ channels reduces Alzheimer disease β-amyloid peptide secretion.

Alzheimer disease (AD), the leading cause of dementia, is characterized by the accumulation of β-amyloid peptides (Aβ) in senile plaques in the brains of affected patients. Many cellular mechanisms are thought to play important roles in the development and progression of AD. Several lines of evidence point to the dysregulation of Ca(2+) homeostasis as underlying aspects of AD pathogenesis. Moreover, direct roles in the regulation of Ca(2+) homeostasis have been demonstrated for proteins encoded by familial AD-linked genes such as PSEN1, PSEN2, and APP, as well as Aβ peptides. Whereas these studies support the hypothesis that disruption of Ca(2+) homeostasis contributes to AD, it is difficult to disentangle the effects of familial AD-linked genes on Aβ production from their effects on Ca(2+) homeostasis. Here, we developed a system in which cellular Ca(2+) homeostasis could be directly manipulated to study the effects on amyloid precursor protein metabolism and Aβ production. We overexpressed stromal interaction molecule 1 (STIM1) and Orai1, the components of the store-operated Ca(2+) entry pathway, to generate cells with constitutive and store depletion-induced Ca(2+) entry. We found striking effects of Ca(2+) entry induced by overexpression of the constitutively active STIM1(D76A) mutant on amyloid precursor protein metabolism. Specifically, constitutive activation of Ca(2+) entry by expression of STIM1(D76A) significantly reduced Aβ secretion. Our results suggest that disruptions in Ca(2+) homeostasis may influence AD pathogenesis directly through the modulation of Aβ production

Loss of Cleavage at β′-Site Contributes to Apparent Increase in β-Amyloid Peptide (Aβ) Secretion by β-Secretase (BACE1)-Glycosylphosphatidylinositol (GPI) Processing of Amyloid Precursor Protein

Several lines of evidence implicate lipid raft microdomains in Alzheimer disease-associated β-amyloid peptide (Aβ) production. Notably, targeting β-secretase (β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1)) exclusively to lipid rafts by the addition of a glycosylphosphatidylinositol (GPI) anchor to its ectodomain has been reported to elevate Aβ secretion. Paradoxically, Aβ secretion is not reduced by the expression of non-raft resident S-palmitoylation-deficient BACE1 (BACE1-4C/A (C474A/C478A/C482A/C485A)). We addressed this apparent discrepancy in raft microdomain-associated BACE1 processing of APP in this study. As previously reported, we found that expression of BACE1-GPI elevated Aβ secretion as compared with wild-type BACE1 (WTBACE1) or BACE1-4C/A. However, this increase occurred without any difference in the levels of APP ectodomain released following BACE1 cleavage (soluble APPβ), arguing against an overall increase in BACE1 processing of APP per se. Further analysis revealed that WTBACE1 cleaves APP at β- and β′-sites, generating +1 and +11 β-C-terminal fragments and secreting intact as well as N-terminally truncated Aβ. In contrast, three different BACE1-GPI chimeras preferentially cleaved APP at the β-site, mainly generating +1 β-C-terminal fragment and secreting intact Aβ. As a consequence, cells expressing BACE1-GPI secreted relatively higher levels of intact Aβ without an increase in BACE1 processing of APP. Markedly reduced cleavage at β′-site exhibited by BACE1-GPI was cell type-independent and insensitive to subcellular localization of APP or the pathogenic KM/NL mutant. We conclude that the apparent elevation in Aβ secretion by BACE1-GPI is mainly attributed to preferential cleavage at the β-site and failure to detect +11 Aβ species secreted by cells expressing WTBACE1