Angèle T. Parent

Blunted Responses to Vasoconstrictors in Mesenteric Vasculature but not in Portal Vein of Spontaneously Hypertensive Rats Treated with Relaxin

Abstract Relaxin (RLX), an ovarian polypeptide hormone that is particularly associated with gestation in viviparous species, has recently been shown to decrease blood pressure in virgin spontaneously hypertensive rats (SHR) upon chronic infusion. In this investigation, vascular reactivity to angiotensin II, arginine-vasopressin, and norepinephrine was studied in the perfused mesenteric artery and isolated portal vein of control and RLX-treated virgin spontaneously hypertensive rats. The latter received an intravenous infusion of 75 ng/hr purified rat RLX for 2 days, whereas the controls were given an equal infusion of saline. All of the animals were then killed and their tissues processed for in vitro study. In the perfused mesenteric artery, the concentration-response curves for arginine-vasopressin and norepinephrine were shifted to the right by a factor of about 2 (P < 0.05 and P < 0.005, respectively) after RLX treatment. In the isolated portal vein, the response to angiotensin II was not affected; the effect of norepinephrine was slightly displaced to the right (increase in EC50) and the maximum response remained unchanged. These results demonstrate that RLX treatment for 42 hr blunted the vascular response to vasoconstrictor agents in the mesenteric vasculature and are consistent with similar observations reported previously in the same tissue of 20-day-old pregnant rats. It is concluded that RLX may be involved in the blunted response to vasoconstrictor agents during gestation in the rat.

A Function for EHD Family Proteins in Unidirectional Retrograde Dendritic Transport of BACE1 and Alzheimer’s Disease Aβ Production

Abnormal accumulation of β-secretase (BACE1) in dystrophic neurites and presynaptic β-amyloid (Aβ) production contribute to Alzheimers disease pathogenesis. Little, however, is known about BACE1 sorting and dynamic transport in neurons. We investigated BACE1 trafficking in hippocampal neurons using live-cell imaging and selective labeling. We report that transport vesicles containing internalized BACE1 in dendrites undergo exclusive retrograde transport toward the soma, whereas they undergo bidirectional transport in axons. Unidirectional dendritic transport requires Eps15-homology-domain-containing (EHD) 1 and 3 protein function. Furthermore, loss of EHD function compromises dynamic axonal transport and overall BACE1 levels in axons. EHD1/3 colocalize with BACE1 and APP β-C-terminal fragments in hippocampal mossy fiber terminals, and their depletion in neurons significantly attenuates Aβ levels. These results demonstrate unidirectional endocytic transport of a dendritic cargo and reveal a role for EHD proteins in neuronal BACE1 transcytosis and Aβ production, processes that are highly relevant for Alzheimers disease.

APP Receptor? To Be or Not To Be

Amyloid precursor protein (APP) and its metabolites play a key role in Alzheimers disease pathogenesis. The idea that APP may function as a receptor has gained momentum based on its structural similarities to type I transmembrane receptors and the identification of putative APP ligands. We review the recent experimental evidence in support of this notion and discuss how this concept is viewed in the field. Specifically, we focus on the structural and functional characteristics of APP as a cell surface receptor, and on its interaction with adaptors and signaling proteins. We also address the importance of APP function as a receptor in Alzheimers disease etiology and discuss how this function might be potentially important for the development of novel therapeutic approaches.

Reduced Alzheimer's Disease β-Amyloid Deposition in Transgenic Mice Expressing S-Palmitoylation-Deficient APH1aL and Nicastrin

Sequential cleavage of amyloid precursor protein by β- and γ-secretases generates β-amyloid peptides (Aβ), which accumulate in the brains of patients with Alzheimers disease. We recently identified S-palmitoylation of two γ-secretase subunits, APH1 and nicastrin. S-Palmitoylation is an essential posttranslational modification for the proper trafficking and function of many neuronal proteins. In cultured cell lines, lack of S-palmitoylation causes instability of nascent APH1 and nicastrin but does not affect γ-secretase processing of amyloid precursor protein. To determine the importance of γ-secretase S-palmitoylation for Aβ deposition in the brain, we generated transgenic mice coexpressing human wild-type or S-palmitoylation-deficient APH1aL and nicastrin in neurons in the forebrain. We found that lack of S-palmitoylation did not impair the ability of APH1aL and nicastrin to form enzymatically active protein complexes with endogenous presenilin 1 and PEN2 or affect the localization of γ-secretase subunits in dendrites and axons of cortical neurons. When we crossed these mice with 85Dbo transgenic mice, which coexpress familial Alzheimers disease-causing amyloid precursor protein and presenilin 1 variants, we found that coexpression of wild-type or mutant APH1aL and nicastrin led to marked stabilization of transgenic presenilin 1 in the brains of double-transgenic mice. Interestingly, we observed a moderate, but significant, reduction in amyloid deposits in the forebrain of mice expressing S-palmitoylation-deficient γ-secretase subunits compared with mice overexpressing wild-type subunits, as well as a reduction in the levels of insoluble Aβ40–42. These results indicate that γ-secretase S-palmitoylation modulates Aβ deposition in the brain.

Localization and regional distribution of p23/TMP21 in the brain.

Sequential processing of amyloid precursor protein by beta- and gamma-secretases generates Alzheimers disease (AD)-associated beta-amyloid peptides. Recently it was reported that the transmembrane protein p23/TMP21 associates with gamma-secretase, and negatively regulates beta-amyloid production. Despite the link between p23 function and AD pathogenesis, the expression of p23 has not been examined in the brain. Here, we describe the detailed immunohistochemical characterization of p23 expression in rodent and human brain. We report that p23 is co-expressed with gamma-secretase subunits in select neuronal cell populations in rodent brain. Interestingly, the steady-state level of p23 in the brain is high during embryonic development and then declines after birth. Furthermore, the steady-state p23 levels are reduced in the brains of individuals with AD. We conclude that p23 is expressed in neurons throughout the brain and the decline in p23 expression during postnatal development may significantly contribute to enhanced beta-amyloid production in the adult brain.

Predominant Expression of Alzheimer’s Disease-Associated BIN1 in Mature Oligodendrocytes and Localization to White Matter Tracts

BackgroundGenome-wide association studies have identified BIN1 within the second most significant susceptibility locus in late-onset Alzheimer’s disease (AD). BIN1 undergoes complex alternative splicing to generate multiple isoforms with diverse functions in multiple cellular processes including endocytosis and membrane remodeling. An increase in BIN1 expression in AD and an interaction between BIN1 and Tau have been reported. However, disparate descriptions of BIN1 expression and localization in the brain previously reported in the literature and the lack of clarity on brain BIN1 isoforms present formidable challenges to our understanding of how genetic variants in BIN1 increase the risk for AD.MethodsIn this study, we analyzed BIN1 mRNA and protein levels in human brain samples from individuals with or without AD. In addition, we characterized the BIN1 expression and isoform diversity in human and rodent tissue by immunohistochemistry and immunoblotting using a panel of BIN1 antibodies.ResultsHere, we report on BIN1 isoform diversity in the human brain and document alterations in the levels of select BIN1 isoforms in individuals with AD. In addition, we report striking BIN1 localization to white matter tracts in rodent and the human brain, and document that the large majority of BIN1 is expressed in mature oligodendrocytes whereas neuronal BIN1 represents a minor fraction. This predominant non-neuronal BIN1 localization contrasts with the strict neuronal expression and presynaptic localization of the BIN1 paralog, Amphiphysin 1. We also observe upregulation of BIN1 at the onset of postnatal myelination in the brain and during differentiation of cultured oligodendrocytes. Finally, we document that the loss of BIN1 significantly correlates with the extent of demyelination in multiple sclerosis lesions.ConclusionOur study provides new insights into the brain distribution and cellular expression of an important risk factor associated with late-onset AD. We propose that efforts to define how genetic variants in BIN1 elevate the risk for AD would behoove to consider BIN1 function in the context of its main expression in mature oligodendrocytes and the potential for a role of BIN1 in the membrane remodeling that accompanies the process of myelination.

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

Backgroundp23 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.ResultsWe generated several lines of transgenic mice expressing human p23 in neurons under the control of Thy-1.2 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.ConclusionsThese 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.

Loss of presenilin function is associated with a selective gain of APP function

Presenilin 1 (PS1) is an essential γ-secretase component, the enzyme responsible for amyloid precursor protein (APP) intramembraneous cleavage. Mutations in PS1 lead to dominant-inheritance of early-onset familial Alzheimer’s disease (FAD). Although expression of FAD-linked PS1 mutations enhances toxic Aβ production, the importance of other APP metabolites and γ-secretase substrates in the etiology of the disease has not been confirmed. We report that neurons expressing FAD-linked PS1 variants or functionally deficient PS1 exhibit enhanced axodendritic outgrowth due to increased levels of APP intracellular C-terminal fragment (APP-CTF). APP expression is required for exuberant neurite outgrowth and hippocampal axonal sprouting observed in knock-in mice expressing FAD-linked PS1 mutation. APP-CTF accumulation initiates CREB signaling cascade through an association of APP-CTF with Gαs protein. We demonstrate that pathological PS1 loss-of-function impinges on neurite formation through a selective APP gain-of-function that could impact on axodendritic connectivity and contribute to aberrant axonal sprouting observed in AD patients. DOI: http://dx.doi.org/10.7554/eLife.15645.001

Regulation of dynamic BACE1 trafficking in neurons

Proteolytic processing of amyloid precursor protein (APP) by β-site APP cleaving enzyme 1 (BACE1) and γ-secretase generates Aβ peptides. APP is constitutively trafficked through the secretory and endocytic pathways in cultured cells and neurons. The identities of cellular organelles and sorting pathways involved in amyloidogenic processing of APP have been extensively investigated. Although a consensus has not yet emerged, there is a general agreement from biochemical and genetic studies on the importance of endocytic APP trafficking for Aβ production. In neurons, APP is trafficked anterogradely along peripheral and central axons, and proteolytically processed during transit. Recent in vivo studies estimated that ~70% of Aβ released in the brain requires ongoing endocytosis, and synaptic activity regulates the vast majority of this endocytosis-dependent Aβ. BACE1 cleavage is thought to be the rate-limiting step in amyloidogenic processing of APP. Little, however, is known about endocytic BACE1 sorting and dynamic transport in neurons. We investigated BACE1 trafficking in cultured hippocampal neurons using live-cell imaging and selective labeling. This approach revealed dynamic neuronal transport of internalized BACE1 in dendrites and axons. BACE1 was transported in vesicles that were positive for markers of recycling endosomes. Dominant-negative expression and siRNA knock-down of proteins involved in endocytic transit revealed that BACE1 is dynamically transported in recycling endosomes and this process significantly contributes to amyloidogenic APP processing. Our results suggest that BACE1 trafficking in neuronal recycling endosomes is likely relevant for presynaptic Aβ production and contributes to Alzheimer’s disease pathogenesis.

Steady‐state increase of cAMP‐response element binding protein, Rac, and PAK signaling in presenilin‐deficient neurons

J. Neurochem.(2008) 104, 1637–1648.