Proteolysis in Neurodegenerative Diseases
5.2. ALZHEIMER’S DISEASE Clinical Features
Alzheimer’s disease is a progressive cognitive disorder that generally appears in the sixth or seventh decade of life and results in a gradual degeneration of memory and cognitive processes. The cognitive deficits incapacitate the AD patients to the point of total dependency on supplemen-tal health care for survival. Although several palliative drug therapies are available for treating AD, none are effective for any length of time (17). Ten percent of people over 65 yr of age are afflicted with AD. With the gradual aging of the American population, it is predicted that a larger fraction of the population will be affected by this disease.
Elevated A` Peptides in Amyloid Plaques of AD Brains
Alzheimer's disease is diagnosed postmortem by typical neuropathology illustrated by the presence of amyloid plaques, especially in hippocampal and cortical brain regions (1–3). The primary component of amyloid plaques is the
`-peptide (or A` peptide) that consists of three peptide forms that differ in their COOH-termini (Fig. 2). These` peptides are comprised of the A`1–40, A`1–
42, and A`1–43 forms that possess 40, 42, or 43 residues of the same primary sequence differing only at their COOH-termini. The A`1–42 form contains two additional amino acids at the COOH-terminus of the A`1–40 peptide form, and the A`1–43 form contains one additional amino acid at its COOH-terminus compared to the A`1–42 form. The three forms of A` are first synthesized as the larger APPs (18–22). Clearly, proteolytic processing of APP is required to generate the smaller A` peptides.
All forms of A` accumulate in AD brains and have been shown in numerous studies to be neurotoxic (23–25). Moreover, there appears to be a preferential elevation of the A`1–42 and A`1–43 peptide forms in AD.
Genetic mutations in the APP gene and in the presenilin 1 and 2 genes have been characterized in transgenic mice and in AD patients (1–3); these studies show a strong relationship between APP and presenilin gene mutations with the observed increases in levels of A`1–42 and A`1–43, amyloid plaque
Fig. 2. Amyloid precursor protein (APP), A` peptides, and secretases. The struc-ture of APP is schematically illustrated. APP contains a signal peptide (SP) sequence, a cysteine-rich region (C-rich), a KPI (kunitz protease inhibitor) domain for APP-751 and APP-770 forms (APP-695 lacks the KPI domain), a transmem-brane domain, and a cytoplasmic COOH-terminal domain. Most importantly, A`
peptides are present within APP near the transmembrane domain. The APP precur-sor undergoes proteolysis by secretases to generate the A` peptides that are known to be neurotoxic, and accumulate in amyloid plaques in AD brains. Proteolytic processing of APP generates three forms of A` peptides of 40, 42, and 43 amino acids in length. These peptide forms possess the same NH2-terminus beginning with Asp; they differ in their COOH-termini, as illustrated. Proteolysis at the
`-secretase site generates the NH2-terminus of A` peptides. Proteolysis at the COOH-termini of A` peptides occurs at a-secretase sites; it is noted that there are three differenta-secretase sites. Proteolysis may also occur within the A` peptide at the _-secretase site, which precludes formation of A` peptides. Although the APP processing sites have been named the `-, a,- and _-secretases, the protease respon-sible for cleavage at these sites have not yet been definitively identified.
formation, and cognitive deficits. These data support the hypothesis of a causal relationship between enhanced A` production and amyloid plaques in AD brains associated with the cognitive deficits that are characteristic of AD.
APP Processing by Secretase Enzymes
All forms of A` peptides are derived from a larger precursor protein, the amyloid precursor protein (APP) (Fig. 2). There are three major forms of APP consisting of 695, 751, and 771 amino acids, which result from alterna-tive splicing of the APP gene product. Each form of APP contains the A`
peptides (18–22). The APP-751 and APP-771 forms include a kunitz protease inhibitor domain.
Proteases, known as ‘‘secretases,’’ produce A` peptides by cleaving APP at specific peptide bonds at or near the NH2- and COOH-termini of the A`
peptide sequences within APP (1,2,18–22). The secretases are categorized according to their specific cleavage sites within APP, which are related to the production of A` peptides. The secretase that cleaves at the NH2 -termi-nal end of A` is known as the `-secretase. The `-secretase is predicted to cleave between Met-?Asp to generate the NH2-terminus of A`. The protease(s) that cleaves at the COOH-termini of A` are known as a-secre-tase(s), which determine whether A`1–40, A`1–42, or A`1–43 are produced.
Production of A`1–40 would require a-secretase cleavage between Val-?Ile.
A`1–42 and A`1–43 production would require a-secretase cleavages between Ala-?Thr and Thr-?Val, respectively. It is not known whether different a-secretases produce the three different forms of A` peptides. However, because specific increases in A`1–42 and A`1–43 occur in AD, compared to lesser changes in A`1–40, it is likely that several a-secretases exist to generate the COOH-termini of the different A` peptides.
In addition to `- and a-secretases, normal cleavage within the A` sequence occurs between Lys-?Leu by _-secretase (1,2). Therefore, _-secretase cleav-age of APP precludes formation of A` peptides.
Genetic studies point to a critical role of the `- and a–secretases to increase the production of A` peptides in AD, especially the extended A`1–
42 and A`1–43 peptide forms. Mutations in the APP gene, which are located near secretase processing sites within APP, are genetically linked to AD in certain families (1–3,26–29). Transgenic mice that overexpress mutant APPs develop brain amyloid plaques, show elevated A` peptide levels in the brain, and display deficits in cognition and memory (1–3,30–32). Moreover, numerous AD-linked genetic mutations in the presenilin 1 and 2 genes enhance the production of A` and favor the elevation of A`1–42 and A`1–43 over A`1–40 in transgenic mice (33,34) and tranfected cell lines (34–36). The selective
increase in A`1–42 and A`1–43 by mutant presenilins suggests that different a-secretases may be responsible for producing the A` peptide forms.
APP Trafficking and Processing in the Secretory Pathway
Neuronal peptides destined for secretion are typically routed to the secre-tory pathway to allow for release of these peptides into the extracellular environment. Studies of the cellular trafficking of APP and its processing are important to define the possible locations of secretases within the cell.
Thus, although the secretases themselves have not been found, numerous studies have established that APP subcellular trafficking and processing occur in the secretory pathway (1–3).
The deduced primary sequence of the human APP cDNA indicates that it possesses an NH2-terminal signal sequence, which serves as a mechanism to route translated proteins to the secretory pathway. The secretion of peptides routed to the secretory pathway are typically stimulated by neuronal recep-tor activation; indeed, muscarinic receprecep-tor stimulation of hippocampal neurons releases A` peptides (37). In addition, APP undergoes axonal transport to nerve terminals (38,39), which is consistent with trafficking of vesicles to axon termi-nals for secretion. Many in vivo studies have provided ample evidence for the trafficking and processing of APP in the secretory pathway.
In vitro studies of APP transfected into cell lines have provided valuable information concerning the subcellular compartments involved in APP trafficking and processing. Investigations of A` peptides, detected by sensitive sandwich enzyme-linked immunosorbent assays (ELISAs), show trafficking of APP in the secretory pathway, where A` peptide production occurs. Evi-dence sugggests that APP processing occurs in the early secretory pathway, including the RER (rough endoplasmic reticulum) and Golgi apparatus, and in post-Golgi vesicles (1,2,40,41). In addition, a high proportion of APP exists in a membrane-bound form and becomes incorporated into the cell membrane (1–3). The APP protein can be internalized from the cell surface to endosomes, where some APP processing may also occur.
These findings predict that APP processing into A` peptides may occur at several locations within the secretory pathway. It is, therefore, logical to predict that the corresponding secretases are present with APP in the secretory pathway. This knowledge is important for consideration of candidate secretases. For example, recent in vitro studies in transfected cells have suggested that caspases may cleave APP to A`, and, thus, caspases have been proposed as candidate secretases (42). However, caspases are present in the cytosol of cells, whereas APP is inaccessible to cytosolic components, because APP is contained within the subcellular organelles of the secretory path-way. Therefore, caspases are not considered to be candidates (43).
Several recent studies reported the identification of a candidate aspartyl protease known as BACE (`-site APP-cleaving enzyme) or Asp2 (44–47) for`-secretase processing of APP. This novel aspartyl protease cDNA clone was obtained by expression cloning of a human embryonic kidney cell cDNA library expressed in HEK293 cells (44), purification and cloning of the human aspartyl protease (45), and bioinformatic approaches to identify aspartyl proteases based on their predicted conserved active site residues (46,47). BACE, or Asp2, increases A` formation when cotransfected with APP in cell lines. Recombinant BACE, or Asp2, has been shown to cleave at the`-secretase site. This enzyme is expressed in the brain, with the highest expression in the pancreas, as well as in the kidney and other tissues. Stud-ies have not yet tested for colocalization of the aspartyl protease with APP, APP-derived intermediates, and A` within the identical cell type and subcellular compartment in vivo. Moreover, it will be important to test these candidate`-secretase enzymes in knockout mice to assess their likelihood as proteases involved in A` formation.
It is predicted that the secretases should be colocalized with APP and A`
peptides in the secretory pathway. It will be most exciting when authentic secretases are established, which is now an area of intense investigation.
Knowledge of the secretases is essential for understanding the proteolytic mechanisms underlying the development of Alzheimer’s disease.
5.3. HUNTINGTON’S DISEASE