The Cell

Cellular Trafficking in Alzheimer’s Disease

According to the Alzheimer’s Association, it is estimated in 2014 that about 5.2 million Americans have Alzheimer’s disease (AD). Of these, about 200,000 are below the age of 65. It is projected that by 2050, unless there is a future effective treatment, there may be about 16 million individuals who are 65 or older who have AD. The annual death rate from AD in the United States is about 500,000. In the year 2014 the estimated cost of AD in the United States is about $214 billion. Because the pathology of AD at the cellular level involves trafficking among different compartments within the affected cell (neuron), AD represents a clinical problem that is illustrative of cellular structure.

Alzheimer’s disease, a disease characterized by progressive loss of memory and the loss of cognition (thinking), is related to the excessive degradation by brain secretases (protease enzymes) of the amyloid precursor protein (APP) to amyloid beta (Αβ) peptides that are neurotoxic. Thus a disease can be caused by an endogenous normally functioning protein that, when modified to an abnormal shape, size, or folding, becomes pathologic similar to other neurodegenerative diseases like Prion disease (Creutzfeldt–Jakob disease in the human; see Chapter 4: Proteins). Aggregation and spread of product of a [alpha]-secretase action damage the brain. This mechanism is pictured in Fig. 2.1 .

Deposition of amyloid (amorphous parenchymal deposits) takes the form of ordered proteinaceous β-sheets (see Chapter 4: Proteins). Amyloid deposition of this type not only occurs in AD but also occurs in Lewy body (abnormal protein aggregates in neurons) dementia, vascular dementia, and Down’s syndrome, all of which are considered to be age-related neurodegenerative diseases.

The APP and its breakdown products (C-terminal fragments and amyloid precursor intracellular domain) are collected in multivesicular bodies within the cell and in secreted exosomes. This trafficking within the cell involves the early endosome that generates multivesicular bodies (multivesicular endosomes) that can either fuse with lysosomes for the degradation of the ingredients or be secreted (exosomes) to the extracellular space. Movements of intracellular particles and secretory contents are captured in Fig. 2.2 .

In the nonamyloidogenic pathway, the intracellular soluble APPα product of a [alpha]-secretase action is not further degraded into products of the actions of β- and γ-secretase that ultimately generate Aβ products leading to their pathological aggregation.

The discovery of a sorting receptor called SORLA (sorting-related receptor with A-type repeats) reveals that it acts to prevent the APP from sorting to the late endosomes where the breakdown product, Aβ, is generated and leads to amyloid deposition. Normal SORLA activity insures that the reactions leading to Alzheimer’s disease do not occur, whereas the mutations in the gene for SORLA to make the sorting receptor less functional or lower in activity can lead to reactions involving the late endosome and the production of A β for amyloid deposition. In Fig. 2.3 are shown the VPS10 (vacuolar protein sorting-10) domain receptors, of which one is the SORLA receptor as well as the overall trafficking of SORLA.

There are two models for the actions of the sorting receptor SORLA that can take trafficking paths to avoid Alzheimer’s disease. In one version, SORLA retains APP in the trans-Golgi network (TGN) preventing the formation of APP homodimers that are the preferred substrates for secretase. In the other model, SORLA binds APP and shuttles between the TGN and the early endosomes, thus reducing APP from amyloidogenic processing in the endosomes. However, the further processing by the secretases (β and γ) finally avails the aggregatable form, Aβ. These models are shown in Fig. 2.4 .

In addition to the deposition of aggregates of Aβ, a second lesion, known as intraneuronal neurofibrillary tangles ( NFT ), is involved in the development of AD. This lesion is an interneuronal aggregation of microtubule-associated Tau proteins (CNS neuronal protein stabilizers of microtubules) that are abnormally modified. AD progresses by virtue of a synergistic relationship between these two types of lesions (Aβ and NFT). A comparison of a normal versus an Alzheimer neuron is shown in Fig. 2.5 .

It is unclear whether cortical atrophy ( Fig. 2.5 ) always occurs in AD because it usually occurs in a different part of the brain (posterior brain cortex). Posterior cortical atrophy is often associated with Lewy body dementia or with Creutzfeldt–Jakob disease .

The Effects of Amyloid Beta (Aβ) on Synapse Function

Aβ clearly has detrimental effects on the functions of the synapse . In addition to its accumulation at the synapse, there are other mechanisms dependent on Aβ that involve the dysfunction of the synapse . Accordingly, Fig. 2.6 summarizes the impact of Aβ on synapse trafficking .

Cell Membrane

As seen in Fig. 1.19, the cells in the body display many different shapes. Therefore in this chapter, we will deal with an ideal model of a typical cell. An example is shown in Fig. 2.7 . The higher eukaryotic cell that contains a distinct membrane-bound nucleus, shown here, contrasts with a prokaryotic cell, a bacterium, or a cyanobacterium, which do not contain membrane-bound organelles and do not have their DNAs in the form of chromosomes. It also contrasts with the eukaryotic yeast cell that contains a rigid cell wall outside the plasma membrane. The cell membrane or the plasma membrane is the outermost layer surrounding the cell ( Fig. 2.7 ) consisting of a double layer of lipids with polar head groups facing the exterior and interior. The lipid bilayer membrane is penetrated with transmembrane proteins with extensions to the outside of the cell and inside to contact the cytoplasm ( Fig. 2.8 ). From the outside layer inward, the polar head groups are ammonium (NH4+), hydrocarbon chain of the fatty acid, and a phosphate group (substituent of glycerol, for one) on the interior side.

The nonpolar stretches of the glycerol-connected fatty acids meet in the middle of the two layers forming the nonpolar region. Each portion of the membrane consisting of a polar group on either end connected to nonpolar stretches in the middle is referred to as a leaflet ( Fig. 2.9 ).

Cholesterol , located between the hydrocarbon components (fatty acid chains), is a critical ingredient of the membrane and is present in about a one-to-one ratio with the phospholipid ( Fig. 2.8 ). A phospholipid consists of one molecule of glycerol substituted by two molecules of fatty acids, a phosphate group, and a polar molecule; examples are phosphatidylcholine, phosphatidylethanolamine, or phosphatidic acid. The polar group attracts water and faces the interior aqueous cytoplasm, while the nonpolar (hydrophobic; repels water) fatty acid tail faces away from the aqueous cytoplasm (see Chapter 9: Lipids). Cholesterol provides some rigidity to the otherwise flexible semipermeable membrane. Cholesterol also enhances the nonpolar solubility of the membrane for entry of nonpolar substances that can dissolve in and permeate the membrane, essentially by free diffusion. There are various groups protruding from the outside surface of the membrane. Glycolipids , for example, may be involved in the specific binding of extracellular proteins to the cell surface. Glycoproteins in the membrane have carbohydrate portions available on the outer cell membrane, each substituent having sugar molecules up to as many as 15 but usually a smaller number ( Fig. 2.8 ). These groups provide the cell’s ability to distinguish self from nonself and are critical for the specific identity of blood types as will be described in later chapters. The cell membrane is a dynamic structure containing many groupings on the outside surface, including extracellular portions of receptors (for subsequent signaling) and channels (serving transporting functions) of all kinds for the recognition of micromolecules and macromolecules. The cell (plasma) membrane permits free diffusion of gases, such as oxygen and carbon dioxide, and small nonpolar substances but it excludes water, charged ions, and sugars for which there exist specific channels in appropriate cells. Thus the interior composition of the cell is protected from the external environment. Table 2.1 summarizes many of the functions of the cell membrane.

Nucleus and Cell Division

Within the cytoplasm, encapsulated by the cell membrane, are found all of the particulate structures that function inside of the cell. The nucleus is the central structure in the cell (about 5 µm or 0.005 mm in diameter) that contains the genetic information of the organism (46 chromosomes in the human, containing information for as many as 32,000 genes involving up to 3 billion base pairs). A model of a typical nucleus is shown in Fig. 2.10 .

The nucleus is the site of gene expression and gene repression under the control of many transcription factors . The nuclear genes express information for all of the cell’s proteins through messenger RNAs except for a small number of proteins encoded in mitochondrial DNA (mtDNA). Some of the proteins encoded by the nucleus ultimately are completed in the cytoplasm and are transported into the mitochondrion.

The nuclear membrane is similar to the plasma membrane and is covered with nuclear pores that are the entries for molecules, such as receptors and other transcription factors. Some molecules are large enough to require transporters or nucleoporins (Nups) to escort these molecules to the nuclear pores; they may ferry the macromolecules through the cytoplasm to docking sites on the nuclear pore complex (a megadalton translocase complex integrated into the nuclear envelope). These pores are also used for egress from the nucleus to the cytoplasm (e.g., export of mRNAs). The nucleus contains cytoplasm, called nucleoplasm . The nucleus contains the chromatin and nucleolus (there may be more than one; Fig. 2.10 ). Chromatin is constituted by the genes. Heterochromatin is the tightly packed or condensed chromatin, whereas euchromatin is the opened form, is less dense, and stringy in appearance. Chromatin needs to be opened to receive messages from transcription factors and to actively transcribe genetic information. Histone proteins are bound to chromosomes, primarily in the nucleosomes that occur periodically on the DNA as shown in Fig. 2.11 .

A nucleus may contain one to four nucleoli ( Fig. 2.10 ). The nucleolus is the main site of ribosomal RNA ( rRNA ) synthesis. It surrounds chromosomes that contain genes for rRNA (there may be hundreds of rRNA genes located in nucleolar-organizing regions at various positions on chromosomes) and the nucleolus does not have a visible membrane. rRNA is synthesized in the nucleolus, translocated to the nucleoplasm, and then through nuclear pores to the cytoplasm where it is assembled into ribosomes as part of the protein synthetic machinery.

The cell cycle dictates the point at which cell division occurs. A diagram of the cell cycle is shown in Fig. 2.12 .

After cell division (mitosis, comprising 2% of the cycle), the smaller daughter cells proceed to the G ₁ interphase (comprising 40% of the cycle), the first growth phase, where the majority of the cells are found. Cells in the G ₁ phase may enter the G ₀ phase where they may await further cell differentiation or may be programmed for cell death (apoptosis). Also during this phase, cells store adenosine triphosphate ( ATP ) and increase in size. Cells entering the S phase (comprising 39% of the cycle) replicate their DNA and then move into the second growth phase, G ₂ (comprising 19% of the cycle). DNA synthesis results in the duplication of chromosomes. The cell is now ready to divide again in mitosis bestowing one set of chromosomes in the daughter cell and retaining one set in the parent. The daughter splits from the parent to form two equal and smaller cells.

The mitotic phase consists of four phases. In prophase the chromosomes become prominent, the nucleolus and nuclear envelope disappear, and the mitotic spindle forms. In metaphase the chromosomes are condensed and highly coiled and are located equidistant from the two poles ( metaphase plate ) and become attached to the newly formed spindle . In anaphase the sister chromatids are now separated. The daughter chromosomes become stationed at opposite poles of the cell. A chromatid is a replicated chromosome having two daughter strands joined by a single centromere (the two strands separate during cell division to become individual chromosomes). A drawing of a chromatid is shown in Fig. 2.13 . In the final telophase, sets of chromosomes are assembled at opposite poles.

A nuclear envelope again forms around each set and division of the cytoplasm ( cytokinesis ) follows. After telophase the cells enter the first growth phase, G ₁ , as described earlier. There are three checkpoints at specific places in the cell cycle ( Fig. 2.12 ). At these points the cell monitors to insure that the functions of the specific phase have been fulfilled. At the metaphase checkpoint , all chromosomes must be attached to the mitotic spindle in order for the cell to continue through the cycle. At the G1 checkpoint , the cell size, availability of nutrients, and the presence of growth factors from other cells are assessed. When the appropriate growth factors from other cells arrive, they generate an increase in the concentration of cyclins (proteins that control progression of cells through the cell cycle) activating cyclin-dependent kinase ( CdK ). CdK phosphorylates and activates S-phase proteins leading to cell division [in cells that are not cycling, the transcription factor, E2F , is normally bound to another protein, the tumor suppressor retinoblastoma protein ( Rb ); the complex inhibits the cell cycle. Arrival of growth factors from other cells activates the cyclin–CdK complex by phosphorylating Rb. As a result, E2F is released and stimulates the production of S-phase proteins]. At the G2 checkpoint , adequate cell size and the successful completion of chromosome replication are evaluated. The cell completes the cycle when all of these measures have been attained successfully. For clarity, cell division is recapitulated from the point of view of the changes occurring within the cell at phases of the cycle in Fig. 2.14 .

There are 23 chromosomes from each parent in the human. X and Y are the sex-determining chromosomes. The remaining chromosomes from each set are autosomes . The chromosomes in metaphase and a diagram of the human chromosomes are shown in Fig. 2.15 .

The nuclear membrane resembles the plasma membrane in that it has two layers. Ribosomes are attached to the cytoplasmic side of the membrane. The two membranes are interrupted at each nucleopore ( nuclear pore ; Fig. 2.10 ). Molecules of molecular weight up to 44,000 can diffuse into the pore, although larger molecules up to 60,000 Da diffuse more slowly through the pore. Molecules above 60,000 Da and 10 nM in diameter, exceeding the pore’s diameter, require energized transport to negotiate the pore. Nuclear transporting factors attach to proteins needing energized transport and hydrolyze ATP to provide the needed energy. Nups are a family of proteins that are involved in the transport of molecules through the nuclear pore, and they are part of the nuclear pore complex . There are multiple copies of about 30 different Nups comprising the nuclear pore complex. Fig. 2.16 is a schematic representation of the nuclear pore complex and its constituents.