Faculty Bibliography
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374
The ubiquitin-proteasome system and the autophagic-lysosomal system in Alzheimer disease
As neurons age, their survival depends on eliminating a growing burden of damaged, potentially toxic proteins and organelles-a capability that declines owing to aging and disease factors. Here, we review the two proteolytic systems principally responsible for protein quality control in neurons and their important contributions to Alzheimer disease pathogenesis. In the first section, the discovery of paired helical filament ubiquitination is described as a backdrop for discussing the importance of the ubiquitin-proteasome system in Alzheimer disease. In the second section, we review the prominent involvement of the lysosomal system beginning with pathological endosomal-lysosomal activation and signaling at the very earliest stages of Alzheimer disease followed by the progressive failure of autophagy. These abnormalities, which result in part from Alzheimer-related genes acting directly on these lysosomal pathways, contribute to the development of each of the Alzheimer neuropathological hallmarks and represent a promising therapeutic target.
PMID: 22908190
ISSN: 2157-1422
CID: 3373282
Neurofilaments at a glance
PMCID:3516374
PMID: 22956720
ISSN: 0021-9533
CID: 179149
Workgroup on NAPA's scientific agenda for a national initiative on Alzheimer's disease
This report outlines a goal-directed scientific agenda for a national initiative to overcome the Alzheimer's disease (AD) crisis. The statement; which reflects the collective views and recommendations of leaders in AD research; is intended to aid the implementation of the National Alzheimer's Project Act (NAPA)'s National Plan to defeat AD. The primary public policy aims of this 10-year scientific agenda are to discover; validate; and develop: (1) a broad range of technologies; tools and algorithms for early detection of people with symptomatic AD; and asymptomatic individuals at elevated risk for AD and other dementias; and (2) a wide range of interventions to preserve and/or restore health and normal neural function; aiming to maintain independent functioning for as long as possible. The long-term scientific public health objectives of this comprehensive plan are to: (1) reduce the number of people with chronic disabling symptoms who will require prolonged care and; eventually; reduce the number of asymptomatic people at elevated risk for AD/dementia; (2) delay the onset of chronic disability for people with AD and other degenerative brain disorders; and (3) lower the cost and burden of care. The plan calls for significant expansion of research programs to identify and validate the cause(s) and pathogenesis of AD; genetic and epigenetic factors that contribute to AD risk; therapeutic targets that affect disease progression; surrogate biomarkers of AD pathobiology; and technologies for early detection of AD.
PMID: 22748940
ISSN: 1552-5279
CID: 3373292
Peripherin is a subunit of peripheral nerve neurofilaments: implications for differential vulnerability of CNS and peripheral nervous system axons
Peripherin, a neuronal intermediate filament protein implicated in neurodegenerative disease, coexists with the neurofilament triplet proteins [neurofilament light (NFL), medium (NFM), and heavy (NFH) chain] but has an unknown function. The earlier peak expression of peripherin than the triplet during brain development and its ability to form homopolymers, unlike the triplet, which are obligate heteropolymers, have supported a widely held view that peripherin and neurofilament triplets form separate filament systems. However, here, we demonstrate that, despite a postnatal decline in expression, peripherin is as abundant as the triplet in the adult PNS and exists in a relatively fixed stoichiometry with these subunits. Peripherin exhibits a distribution pattern identical to those of triplet proteins in sciatic axons and colocalizes with NFL on single neurofilaments by immunogold electron microscopy. Peripherin also coassembles into a single network of filaments containing NFL, NFM, and NFH with and without alpha-internexin in quadruple- or quintuple-transfected SW13vim(-) cells. Genetically deleting NFL in mice dramatically reduces peripherin content in sciatic axons. Moreover, peripherin mutations has been shown to disrupt the neurofilament network in transfected SW13vim(-) cells. These data show that peripherin and the neurofilament proteins are functionally interdependent. The results strongly support the view that, rather than forming an independent structure, peripherin is a subunit of neurofilaments in the adult PNS. Our findings provide a basis for its close relationship with neurofilaments in PNS diseases associated with neurofilament accumulation.
PMCID:3405552
PMID: 22723690
ISSN: 0270-6474
CID: 170430
Calpastatin modulates APP processing in the brains of beta-amyloid depositing but not wild-type mice
We report that neuronal overexpression of the endogenous inhibitor of calpains, calpastatin (CAST), in a mouse model of human Alzheimer's disease (AD) beta-amyloidosis, the APP23 mouse, reduces beta-amyloid (Abeta) pathology and Abeta levels when comparing aged, double transgenic (tg) APP23/CAST with APP23 mice. Concurrent with Abeta plaque deposition, aged APP23/CAST mice show a decrease in the steady-state brain levels of the amyloid precursor protein (APP) and APP C-terminal fragments (CTFs) when compared with APP23 mice. This CAST-dependent decrease in APP metabolite levels was not observed in single tg CAST mice expressing endogenous APP or in younger, Abeta plaque predepositing APP23/CAST mice. We also determined that the CAST-mediated inhibition of calpain activity in the brain is greater in the CAST mice with Abeta pathology than in non-APP tg mice, as demonstrated by a decrease in calpain-mediated cytoskeleton protein cleavage. Moreover, aged APP23/CAST mice have reduced extracellular signal-regulated kinase 1/2 (ERK1/2) activity and tau phosphorylation when compared with APP23 mice. In summary, in vivo calpain inhibition mediated by CAST transgene expression reduces Abeta pathology in APP23 mice, with our findings further suggesting that APP metabolism is modified by CAST overexpression as the mice develop Abeta pathology. Our results indicate that the calpain system in neurons is more responsive to CAST inhibition under conditions of Abeta pathology, suggesting that in the disease state neurons may be more sensitive to the therapeutic use of calpain inhibitors.
PMCID:3318946
PMID: 22206846
ISSN: 0197-4580
CID: 164336
Guidelines for the use and interpretation of assays for monitoring autophagy [Guideline]
In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
PMCID:3404883
PMID: 22966490
ISSN: 1554-8627
CID: 181862
Tissue processing prior to analysis of Alzheimer's disease associated proteins and metabolites, including abeta
Amyloid-containing tissue, whether from human patients or an animal model of a disease, is typically characterized by various biochemical and immunohistochemical techniques, many of which are described in detail in this volume. In this chapter, we describe a straightforward technique for the homogenization of tissue prior to these analyses. The technique is particularly well suited for performing a large number of different biochemical analyses on a single mouse brain hemisphere. Starting with this homogenate multiple characterizations can be done, including western blot analysis and isolation of membrane-associated proteins, both of which are described here. Additional analyses can readily be performed on the tissue homogenate, including the ELISA quantitation of Abeta in the brain of a transgenic mouse model of beta-amyloid deposition. The ELISA technique is described in detail in Chapter 34 .
PMID: 22528111
ISSN: 1064-3745
CID: 166105
Autophagy failure in Alzheimer.s disease and lysosomal storage disorders: A common pathway to neurodegeneration?
pp. 237-257
ISBN: 9789814350457
CID: 2171242
Abeta measurement by enzyme-linked immunosorbent assay
The neuritic plaque in the brain of Alzheimer's disease patients consists of an amyloid composed primarily of Abeta, an approximately 4-kDa peptide derived from the amyloid precursor protein. Multiple lines of evidence suggest that Abeta plays a key role in the pathogenesis of the disease, and potential treatments that target Abeta production and/or Abeta accumulation in the brain as beta-amyloid are being aggressively pursued. Methods to quantitate the Abeta peptide are, therefore, invaluable to most studies aimed at a better understanding of the molecular etiology of the disease and in assessing potential therapeutics. Although other techniques have been used to measure Abeta in the brains of AD patients and beta-amyloid-depositing transgenic mice, the enzyme-linked immunosorbent assay (ELISA) is one of the most commonly used, reliable, and sensitive methods for quantitating the Abeta peptide. Here we describe methods for the recovery of both soluble and deposited Abeta from brain tissue and the subsequent quantitation of the peptide by sandwich ELISA.
PMID: 22528112
ISSN: 1064-3745
CID: 166104
The C-Terminal Domains of NF-H and NF-M Subunits Maintain Axonal Neurofilament Content by Blocking Turnover of the Stationary Neurofilament Network
Newly synthesized neurofilaments or protofilaments are incorporated into a highly stable stationary cytoskeleton network as they are transported along axons. Although the heavily phosphorylated carboxyl-terminal tail domains of the heavy and medium neurofilament (NF) subunits have been proposed to contribute to this process and particularly to stability of this structure, their function is still obscure. Here we show in NF-H/M tail deletion [NF-(H/M)(tailDelta)] mice that the deletion of both of these domains selectively lowers NF levels 3-6 fold along optic axons without altering either rates of subunit synthesis or the rate of slow axonal transport of NF. Pulse labeling studies carried out over 90 days revealed a significantly faster rate of disappearance of NF from the stationary NF network of optic axons in NF-(H/M)(tailDelta) mice. Faster NF disappearance was accompanied by elevated levels of NF-L proteolytic fragments in NF-(H/M)(tailDelta) axons. We conclude that NF-H and NF-M C-terminal domains do not normally regulate NF transport rates as previously proposed, but instead increase the proteolytic resistance of NF, thereby stabilizing the stationary neurofilament cytoskeleton along axons.
PMCID:3448626
PMID: 23028520
ISSN: 1932-6203
CID: 179098
