In order to make progress past the anatomical level of observation, and distinguish between different kinds of neurological disorders, greater resolution of brain tissue was needed. That was provided by the light microscope. Invented by the Dutch lens-maker Anton van Leeuwenhoek (1632–1723) and improved upon by the English scientist and inventor Robert Hooke (1635–1703). This device enables the user to observe cells, and their organelles, and other macromolecular substructures. Contrast is important, and advances in the field have occurred whenever new and improved dyes have been discovered that make the macromolecular structures of interest stand out against the cellular background. The amount of detail made visible by a light microscope is limited by the wavelength of the probe being used. When used with the right dyes the light microscope can resolve structures down to about 0.2 μm, or 200 nm (half the 400 nm nominal wavelength of visible light).

The light microscope is the primary tool of the pathologist. This field has its modern beginnings in the pioneering studies of Rudolf Virchow (1821–1902) in the mid-nineteenth century. Virchow is regarded as the father of modern pathology. In 1854, he applied the term “amyloid” to describe unusual deposits found in the liver, the spleen, and other organs and tissues of people who had succumbed to illnesses ranging from tuberculosis to syphilis. These deposits were initially thought to be fatty or waxy in nature; the term amyloid had been coined to a few years earlier, in 1838, by the botanist Mathias Schleiden in his description of starch and cellulose. The correct identity that these deposits were proteinaceous was made a few years after Virchow’s initial discoveries.

The first steps in discriminating between the different kinds of neurological disorders were taken by Charcot who combined careful clinical observations with anatomical studies and tissue pathology. He was the first to describe amyotrophic lateral sclerosis (ALS). In his lectures delivered at the Hôpital de la Salpêtrière in Paris, he distinguished between upper and lower motor neurons and began the process of separating the different motor diseases from one another. In the United States, this disorder is referred to as Lou Gehrig’s disease after the famous baseball player who died from it in 1941, but on the Continent and elsewhere it is known as Charcot’s disease.

The next and arguably the most famous in a series of landmark events was the 1906 presentation by Alzheimer (Fig. 1.2). In it, he described his postmortem analysis of brain tissue taken from Auguste Deter, a 51-year-old patient (in 1901) suffering from early-onset dementia. Using a light microscope, and utilizing the newly developed Golgi silver staining techniques (see Appendix 1), Alzheimer found that the tissue was permeated with extracellular deposits that he called “miliary foci” and are now referred to as extracellular plaques. He also observed that the cells themselves contained dense bundles of fibrils. He then made the conceptual leap that these deposits were in some manner causally connected to the patient’s dementia.

Alzheimer’s findings were not unique to the dementia he was exploring, or the story would end there. Instead, his discoveries were preceded by those of Andrew Pick (1851–1924) who reported in 1892 that deposits were present in brain tissue of deceased patients suffering from senile dementia. These aggregates are now known as Pick bodies, and these deposits along with an extreme atrophy in the frontal and temporal regions of the brain characterize the dementia. Clinically the disorder observed by Pick differs from Alzheimer’s disease. In the former case, social and personality deficits predominate in the early stages whereas in the latter instance memory loss occurs first. A few years after Alzheimer’s report, in 1912, Frederic Lewy (1885–1950) described the presence of intracellular deposits (now known as Lewy bodies ) in the brains of patients with Parkinson’s disease. Thus, it was becoming clear that deposits of some sort of protein are associated with a large number of different diseases of the brain.

The question then naturally arose as to whether the brain deposits were the same or similar to those seen elsewhere in the body by Virchow and his successors. That question was answered in the affirmative by the development of a test specific for amyloid in 1927. In that year, Paul Divry (1889–1967) and Marcel Florkin (1900–1979) published results of their studies of various body amyloid tissues stained with a dye called Congo red and viewed using polarized light. When that is done, the amyloid tissue exhibits an apple-green birefringence. In a second 1927 paper, Divry showed that the same apple-green birefringence can be seen for extracellular plaques and again in 1934 for the intracellular tangles. The dye, Congo red, used by Divry originated in the textile industry where it had been invented in 1883 and was given that mysterious-sounding name Congo red as a marketing ploy. Shortly thereafter it was tried as a potential histological stain for tissues. Although it is no longer used to stain textiles, it is still widely used to detect the presence of amyloid in the manner established by Divry, that is, in conjunction with polarized light and the revealing of apple-green birefringence patterns (Fig. 1.3).

Light microscopes are not able to reveal the underlying structure of amyloids. They simply do not have sufficient resolution, but an electron microscope does—about three orders of magnitude greater resolution than a light microscope. The existence of birefringence patterns for these deposits makes it likely that their underlying components are arranged in an orderly fashion rather than in an amorphous manner as most people thought. In 1959, Cohen and Calkins used an electron microscope (Appendix 2) to examine the submicroscopic structure of amyloids taken from different organs of the body (spleen, liver, and kidney). They found that amyloids do indeed possess an orderly substructure. The fibrils are straight and rigid—from 75 to 120 Å in width and vary in length from 1000 to 16,000 Å (Fig. 1.4). The fibrils, in turn, are composed of protofibrils; these filamentous subunits range from 25 to 35 Å in diameter. Two or more of these units, twined about one another, form the fibril. Finally and significantly, they observed a similar structural arrangement for all the amyloids they examined.

To summarize, by 1960 it had become apparent that amyloids deposits were associated in some as yet to be determined manner with a diverse collection of illnesses of the body and mind. These aggregates were present in extracellular spaces and within cells of various tissues and organs. Amyloids are proteinaceous aggregates characterized by: (1) birefringence patterns when stained with Congo red and viewed under polarizing light microscope conditions, and (2) composed of long-twisted fibrillar structures when examined using an electron microscope.

As is the case with any major findings in science, answering one set of questions leads to the next set of perhaps even better questions. In the case of the amyloids, the first question that arises follows from the striking similarity of the structures observed in different parts of the body. The question is: are all amyloid deposit s generated by the same protein or are amyloid deposits generated by different proteins perhaps specific to the tissues or disease states to which they are associated?

To answer this type of question, the primary amino acid sequences of the amyloidogenic proteins needed to be determined. This was accomplished for the first time in 1971 and then over the followed couple of years by several research groups starting with efforts headed by George Glenner and Earl Benditt. These investigators found that the proteins were not the same, but rather each one amyloid protein was unique to the disease associated with their presence. The primary structure, that is, the amino acid sequences, of the amyloidogenic proteins found in different tissues and organs had little resemblance to one another; they normally fold into a diverse set of 3D structures, and unlike the amyloids are soluble. The diverse set of amyloidogenic proteins, their precursors, and the disorders associated with them are summarized in Table 1.1.

The deposits of waxy materials puzzled over by Virchow in the 1850s occurred in the spleen, liver, and kidneys. They were most likely due to what is today called AA amyloidosis involving buildup of amyloid A (AA) protein over time as a consequence of long-lasting infections and chronic inflammation produced, for example, by tuberculosis and rheumatoid arthritis. Amyloid A protein deposits are derived from an overproduction of serum amyloid A (SAA), an acute phase protein serving as a precursor for the AA protein. These buildups occur most often in the heart but can occur in other tissues and organs as well as observed by Virchow.

In systemic amyloid diseases such as AA amyloidosis discussed in the previous paragraph, the amyloids collect in multiple organs and tissues of the body. In another disease of this type, immunoglobulin light chain amyloidosis (AL), B-cells secrete light chain peptides that circulate through the body and collect in the kidneys and also in the heart and liver leading to organ failure and death. Light chain amyloidosis is associated with B-cell myelomas. In contrast to the abovementioned systemic amyloidoses, there are a number of diseases in which amyloid deposit ion is specific to a single organ or tissues of the body. An example of this class of amyloids diseases is Type 2 diabetes in which aggregates of islet amyloid polypeptide (IAPP, amylin) form in the pancreas. These islet amyloid deposits of IAPP are believed to be a major contributor to β-cell failure.

As indicated in Table 1.1 the amyloidogenic proteins are anomalous deposits built from nonamyloidogenic precursors or from fragments thereof. In order to probe further what has occurred to produce the anomalous deposits one has to examine the atomic level structure of the proteins. This requires using more powerful techniques than light or electron microscopy. The paramount experimental techniques for exploring protein structure at atomic level of detail are X-ray crystallography and nuclear magnetic resonance (NMR). These tools, and especially X-ray crystallography, were used to explore the composition of the deposits in the later 1960s and produced a breakthrough set of findings. Specifically, two sets of results were reported—one set by Eanes and Glenner in 1968 and the other by Bonar, Cohen, and Skinner in 1969. In brief, these groups reported that the amyloidogenic proteins have folded into three-dimensional forms quite different from their normal ones. In both studies, the amino acid residues are folded back and forth to form a series of β-pleated sheets. The protein chains in this configuration were then stacked in a perpendicular orientation one on top of the other along the fibril axis. A modern rendering of this type of structure is presented in Fig. 1.5.

Transthyretin (TTR) is a carrier protein that transports thyroxine (T4) and retinol (vitamin A). In its amyloidogenic ATTR form, it is associated with three diseases in the elderly—senile systemic amyloidosis (SSA), familial amyloid polyneuropathy (FAP), and familial amyloid cardiopathy. This protein became the subject of a number of pioneering studies of amyloid formation. One of these was that while TTR was normally soluble, if the pH conditions in their environment were lowered, the proteins partially unfolded and then refolded into an alternate 3D conformation. In that shape, the TTR proteins became aggregation-prone, insoluble, and were capable of forming amyloid fibrils . This type of result demonstrating that the biochemical and biophysical environment has as influence on how proteins fold will be examined in greater detail in the next two chapters. Suffice it to say that at the time the results solidified the main conclusion from both the biophysics and biochemistry—normally soluble proteins have folded into a conformation different from their functionally expected one resulting in the formation of insoluble (and detergent-resistant) amyloids.

Thus, an amyloid is a material satisfying three criteria. It:

The linkage between these levels is a tight one—it was shown by the Glenner group in 1972 that the atomic level, beta-sheet arrangement is indeed responsible for the properties revealed by Congo red staining.

That Alzheimer’s disease is not the only form of senile dementia has been realized still more recently with the emergence of frontotemporal lobar dementia (FTLD) as the second most prominent form of dementia in the young after Alzheimer’s disease. In Alzheimer’s disease, cognitive functions decline, beginning with learning and episodic memory (current and past experiences) and progressing to thinking, loss of language, and further decline in memory. In FTLD/FTD the first, clinically relevant signs involve changes in behavior and personality. As depicted in Fig. 1.6, each class of disease is associated with a specific region of the brain, each giving rise to distinct sets of clinical symptoms.

One of the first sets of goals in the field was to characterize the extra- and intracellular deposits of proteins associated with the neurodegenerative diseases. As indicated in Fig. 1.6 extracellular and especially intracellular deposits are present not just in Alzheimer’s disease and Parkinson’s disease but in all the others as well. First, there was a need to identify the composition of the extracellular plaques and neurofibrillary tangles of Alzheimer’s disease, the Lewy bodies seen in Parkinson’s disease, and the others. Secondly, there was a need to tie the constituents whenever possible to genetic mutations in familial cases of the disease, or to other abnormalities, thereby providing evidence that these deposits are causally connected to the neurodegeneration. These efforts required the concerted application of an ensemble of newly developed biochemical and genetics techniques.