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The transmembrane protein amyloid-[beta] precursor protein (APP) and the vesicle-associated protein c-Jun N[H.sub.2]-terminal kinase-interacting protein-1 (JIP-1) are transported into axons by kinesin-1. Both proteins may bind to kinesin-1 directly and can be transported separately. Because JIP-1 and APP can interact, kinesin-1 may recruit them as a complex, enabling their cotransport. In this study, we tested whether APP and JIP-1 are transported together or separately on different vesicles. We found that, within the cellular context, JIP-1 preferentially interacts with [Thr.sup.668]-phosphorylated APP (pAPP), compared with nonphosphorylated APP. In neurons, JIP-1 colocalizes with vesicles containing pAPP and is excluded from those containing nonphosphorylated APP. The accumulation of JIP-1 and pAPP in neurites requires kinesin-1, and the expression of a phosphomimetic APP mutant increases JIP-1 transport. Down-regulation of JIP-1 by small interfering RNA specifically impairs transport of pAPP, with no effect on the trafficking of nonphosphorylated APP. These results indicate that the phosphorylation of APP regulates the formation of a pAPP-JIP-1 complex that accumulates in neurites independent of nonphosphorylated APP.

Background: Amyotrophic lateral sclerosis (ALS), a debilitating neurodegenerative disorder of the motor neurons, leads to the disorganization of the neurofilament (NF) cytoskeleton and - ultimately - the deterioration of the neuromuscular junction. Some familial cases of ALS are caused by mutated FUS, TDP-43 or SOD1; it is thought that the mutated proteins inflict pathology either by gain or loss of function. The proper function of the neuromuscular junction requires sAPP, a soluble proteolytic fragment of the amyloid-β precursor protein (APP) - a transmembrane protein implicated in the pathology of Alzheimer's disease (AD). Whether sAPP, FUS, TDP-43 and SOD1 are mechanistically linked in a common pathway deregulated in both AD and ALS is not known. Summary: We show that sAPP, TDP-43, FUS and SOD1 are transported to neurite terminals by a mechanism that involves endoplasmic reticulum (ER)-like tubules and requires peripherin NFs. The transport of these proteins, and the translocation of the ER protein reticulon 4 (Rtn4) into neurites was studied in CAD cells, a brainstem-derived neuronal cell line highly relevant to AD and ALS. We show that a significant fraction of sAPP is generated in the soma and accumulates in a juxtanuclear ER subdomain. In neurites, sAPP localizes to Rtn4-positive ER-like tubules that extend from the soma into the growth cone and colocalizes with peripherin NFs. Knocking down peripherin disrupts the NF network and diminishes the accumulation of sAPP, TDP-43, FUS, SOD1 and Rtn4 at terminals. Key Messages: We propose that the impediment of a common, ER-mediated mechanism of transport of sAPP, TDP-43, FUS and SOD1, caused by a disrupted NF network, could be part of the mechanisms leading to AD and ALS. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background and Objective: The pathology of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder affecting motor neurons, comprises aberrant accumulations of neurofilaments; mutations in the peripherin subunit of neurofilaments have been identified in some forms of ALS. Recently, the amyloid-β precursor protein (APP), a key element for the pathology of Alzheimer's disease (AD), was linked to ALS. Here, we provide evidence that the generation of the N-terminal fragment of APP, sAPP, relies on peripherin neurofilaments. This finding could relate to a novel molecular mechanism dysregulated in ALS and/or AD. Methods and Results: The production and the fate of sAPP were studied with the brainstem-derived, neuronal cell line, CAD, which expresses endogenous peripherin. We show that sAPP and C-terminal fragments (CTF) are generated to a large extent in the neuronal soma. We find that sAPP, but not CTF, associates with filamentous structures that delineate the nuclear lamina, extend to the cell periphery and immunostain for peripherin. The depletion of peripherin with siRNA eliminates the filamentous immunostaining of sAPP. Conclusion: Our results indicate that a fraction of APP is cleaved by β-secretase in the soma and that the generated sAPP becomes associated with perinuclear peripherin neurofilaments. These findings link the metabolism of APP - which is dysregulated in AD - to the organization of neurofilaments - which is abnormal in ALS - and suggest a possible crosstalk/overlap between the molecular mechanisms of these diseases. Copyright © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background and Objective: Could a normal - but persistent - stress response to impeded axonal transport lead to late-onset Alzheimer's disease (AD)? Our results offer an affirmative answer, suggesting a mechanism for the abnormal production of amyloid-β (Aβ), triggered by the slowed axonal transport at old age. We hypothesize that Aβ precursor protein (APP) is a sensor at the endoplasmic reticulum (ER) that detects, and signals to the nucleus, abnormalities in axonal transport. When persistently activated, due to chronically slowed-down transport, this signaling pathway leads to accumulation of Aβ within the ER. Methods and Results: We tested this hypothesis with the neuronal cell line CAD. We show that, normally, a fraction of APP is transported into neurites by recruiting kinesin-1 via the adaptor protein, Fe65. Under conditions that block kinesin-1-dependent transport, APP, Fe65 and kinesin-1 accumulate in the soma, and form a complex at the ER. This complex recruits active c-Jun N-terminal kinase (JNK), which phosphorylates APP at Thr668. This phosphorylation increases the cleavage of APP by the amyloidogenic pathway, which generates Aβ within the ER lumen, and releases Fe65 into the cytoplasm. Part of the released Fe65 translocates into the nucleus, likely to initiate a gene transcription response to arrested transport. Prolonged arrest of kinesin-1-dependent transport could thus lead to accumulation and oligomerization of Aβ in the ER. Conclusion: These results support a model where the APP:Fe65 complex is a sensor at the ER for detecting the increased level of kinesin-1 caused by halted transport, which signals to the nucleus, while concomitantly generating an oligomerization-prone pool of Aβ in the ER. Our hypothesis could thus explain a pathogenic mechanism in AD. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The amyloid-β (Aβ) precursor protein (APP), a transmembrane protein that undergoes proteolytic cleavage into defined fragments, has been implicated in axonal transport. The proposed role of APP as a vesicle receptor for the microtubule motor kinesin-1 has relevance for the pathogenesis of Alzheimer's disease. Nevertheless, this function, which relies on the transport to the cell periphery of full-length APP rather than its cleavage fragments, remains controversial. Other proposed functions of APP, such as regulating transcription, neurogenesis, cell movement, or neurite growth also rely on APP's presence as a full-length protein at the cell surface, implying that APP cleavage occurs after its transport to the cell periphery. To test this hypothesis, we mapped the localization of various APP epitopes in neurons in culture and in the mouse brain. Surprisingly, epitopes from the N-terminal, C-terminal, and central (Aβ) domains of APP each showed a distinct distribution throughout the cell and rarely colocalized. Within neurites, these epitopes were localized to distinct transport vesicles that associated with different sets of microtubules and, occasionally, actin filaments. C-terminal APP fragments were preferentially transported into neurites as phosphorylated forms, entered the lamellipodium and filopodia of growth cones, and concentrated in regions of growth cone turning and advancement (unlike the N-terminal and Aβ fragments). We conclude that, under normal conditions, the proteolytic cleavage of APP primarily occurs before its sorting into axonal transport vesicles and the cleaved fragments segregate into separate vesicle populations that reach different destinations, and thus have different functions. [ABSTRACT FROM AUTHOR]

The transmembrane protein amyloid-β precursor protein (APP) and the vesicle-associated protein c-Jun NH2-terminal kinase-interacting protein-1 (JIP-1) are transported into axons by kinesin-1. Both proteins may bind to kinesin-1 directly and can be transported separately. Because JIP-1 and APP can interact, kinesin-1 may recruit them as a complex, enabling their cotransport. In this study, we tested whether APP and JIP-1 are transported together or separately on different vesicles. We found that, within the cellular context, JIP-1 preferentially interacts with Thr668-phosphorylated APP (pAPP), compared with nonphosphorylated APR In neurons, JIP-1 colocalizes with vesicles containing pAPP and is excluded from those containing nonphosphorylated APP. The accumulation of JIP-1 and pAPP in neurites requires kinesin-1, and the expression of a phosphomimetic APP mutant increases JIP-1 transport. Down-regulation of JIP-1 by small interfering RNA specifically impairs transport of pAPP, with no effect on the trafficking of nonphosphorylated APP. These results indicate that the phosphorylation of APP regulates the formation of a pAPP-JIP-1 complex that accumulates in neurites independent of nonphosphorylated APP. [ABSTRACT FROM AUTHOR]

Abnormal phosphorylation of amyloid-β precursor protein (APP) is a pathologic feature of Alzheimer's disease. To begin to understand the mechanism of APP phosphorylation, we studied this process in differentiating neurons under normal physiological conditions. We found that c-Jun NH2-terminal kinase (JNK), not cyclin-dependent kinase 5, is required for APP phosphorylation, leading to localized accumulation of phosphorylated APP (pAPP) in neurites. We show that JNK-interacting protein-3 (JIP-3), a JNK scaffolding protein that does not bind APP, selectively increases APP phosphorylation, accumulation of pAPP into processes, and stimulates process extension in both neurons and COS-1 cells. Downregulation of JIP-3 by small interfering RNA impairs neurite extension and reduces the amount of localized pAPP. Finally, whereas stress-activated JNK generates pAPP only in the cell body, concomitant expression of JIP-3 restores pAPP accumulation into neurites. Thus, APP phosphorylation, transport of the generated pAPP into neurites, and neurite extension are interdependent processes regulated by JIP-3/JNK, in a pathway distinct from stress-activated JNK signaling. [ABSTRACT FROM AUTHOR]

Phosphorylation of amyloid-beta precursor protein (APP) at Thr(668) is a normal process linked to neurite extension and anterograde transport of vesicular cargo. By contrast, increased phosphorylation of APP is a pathological trait of Alzheimer's disease. APP is overexpressed in Down's syndrome, a condition that occasionally leads to increased APP phosphorylation, in cultured cells. Whether phosphorylation of APP in normal versus high APP conditions occurs by similar or distinct signaling pathways is not known. Here, we addressed this problem using brainstem-derived neurons (CAD cells). CAD cells that ectopically overexpress APP frequently show features of degenerating neurons. We found that, in degenerating cells, APP is hyperphosphorylated and colocalizes with early endosomes. By contrast, in normal CAD cells, phosphorylated APP (pAPP) is excluded from endosomes, and localizes to the Golgi apparatus and to transport vesicles within the neurites. Whereas the neuritic APP is phosphorylated by c-Jun NH(2)-terminal kinase through a pathway that is modulated by glycogen synthase kinase 3beta, the endosomal pAPP in degenerated CAD cells results from activation of cyclin-dependent kinase 5. Additional signaling pathways, leading to APP phosphorylation, become active during stress and mitosis. We conclude that distinct pathways of APP phosphorylation operate in proliferating, differentiating, stressed, and degenerating neurons.

Our goal is to understand the pathogenesis of amyloid-beta (Abeta) deposition in the Alzheimer's disease (AD) brain. We established a cell culture system where central nervous system-derived neuronal cells (CAD cells) produce and accumulate within their processes large amounts of Abeta peptide, similar to what is believed to occur in brain neurons, in the initial phases of AD. Using this system, we show that accumulation of Abeta begins within neurites, prior to any detectable signs of neurodegeneration or abnormal vesicular transport. Neuritic accumulation of Abeta is restricted to a small population of neighboring cells that express normal levels of amyloid-beta precursor protein (APP) but show redistribution of BACE1 to the processes, where it colocalizes with Abeta and markers of late endosomes. Consistently, cells that accumulate Abeta appear in isolated islets, suggesting their clonal origin from a few cells that show a propensity to accumulate Abeta. These results suggest that Abeta accumulation is initiated in a small number of neurons by intracellular determinants that alter APP metabolism and lead to Abeta deposition and neurodegeneration. CAD cells appear to recapitulate the biochemical processes leading to Abeta deposition, thus providing an experimental in vitro system for studying the molecular pathobiology of AD.

Abnormal phosphorylation of amyloid-beta precursor protein (APP) is a pathologic feature of Alzheimer's disease. To begin to understand the mechanism of APP phosphorylation, we studied this process in differentiating neurons under normal physiological conditions. We found that c-Jun NH2-terminal kinase (JNK), not cyclin-dependent kinase 5, is required for APP phosphorylation, leading to localized accumulation of phosphorylated APP (pAPP) in neurites. We show that JNK-interacting protein-3 (JIP-3), a JNK scaffolding protein that does not bind APP, selectively increases APP phosphorylation, accumulation of pAPP into processes, and stimulates process extension in both neurons and COS-1 cells. Downregulation of JIP-3 by small interfering RNA impairs neurite extension and reduces the amount of localized pAPP. Finally, whereas stress-activated JNK generates pAPP only in the cell body, concomitant expression of JIP-3 restores pAPP accumulation into neurites. Thus, APP phosphorylation, transport of the generated pAPP into neurites, and neurite extension are interdependent processes regulated by JIP-3/JNK, in a pathway distinct from stress-activated JNK signaling.

Beta-amyloid precursor protein (APP) is implicated in the pathobiology of Alzheimer's disease (AD). To gain insight into its function, we have investigated the proteolytic processing and post-translational modification of APP in relation to its intracellular traffic and localization. The proteolytic processing that generates the amyloid beta-peptide (Abeta) also releases into the cytoplasm the carboxy-terminal fragment of APP, Cgamma. Using the catecholaminergic cell line, CAD, and an antibody to a form of APP that is phosphorylated at Thr668 (pAPP; numbering for APP695), we show that a phosphorylated, carboxy-terminal fragment of APP, probably Cgamma, is present in the nucleus, where it localizes to subnuclear particles. The labeling with anti-pAPP antibody co-localizes with proteins that define the splicing factor compartment (SFC) [e.g. the small nuclear ribonucleoprotein (snRNP), U2B, and serine/arginine-rich (SR) proteins], but is excluded from the coiled bodies and the gems. This distribution of pAPP epitopes was found in CAD cells independent of their state of differentiation, as well as in primary cortical neurons, epithelial cells and fibroblasts. We further show that exogenously expressed Cgamma becomes phosphorylated, and distributes throughout the cell. A fraction of this Cgamma is translocated into the nucleus, where it co-localizes with endogenous pAPP epitopes. Finally, we show that the APP binding, scaffolding protein, Fe65 co-localizes with pAPP epitopes and with expressed Cgamma at intranuclear speckles. These results suggest that phosphorylated Cgamma accumulates at the SFC. Thus, APP may play a role in pre-mRNA splicing, and Fe65 and APP phosphorylation may regulate this function.