John Q. Trojanowski: neuropathology icon

John Q. Trojanowski, M.D., Ph.D., was a giant in the field of neurodegenerative diseases, a clinical and experimental neuropathologist of remarkable stature, a colleague to many, and a friend to even more. John passed away on February 8th, 2022, with his lifelong partner and wife, Virginia M.-Y. Lee, Ph.D., and other family by his side following complications from a debilitating and paralyzing fall and spinal cord injury shortly after his 75th birthday. John was a larger-than-life leader in neurodegenerative disease research with a probing intellect, limitless enthusiasm and energy, and ever-present personality. Often described as a “rock-star,” his physically towering figure and flowing hair (Fig. 1) was counterbalanced by his penchant for fanny packs. His was an extraordinary life.

Fig. 1

John Q. Trojanowski. Photo credit—Scott H. Spitzer, Office of University Communications, University of Pennsylvania

Prescient beginnings

Leonard Jarrett was chair of the Department of Pathology and Laboratory Medicine at the University of Pennsylvania from 1980 to 1998 where he oversaw a tremendous time of growth and development. Jarrett has said that the most important thing he did as chair was to hire John Q. Trojanowski and his partner, Virginia M.-Y. Lee, to the faculty in 1981 (Fig. 2). While still a trainee, John had already written his first successful NIH R01 application to use lectins for neuroanatomic and neuropathologic studies before officially joining the academic ranks. John soon realized the value in maintaining a brain bank, resolute in his conviction that studying human tissue can lead to fundamental insights into disease mechanisms. However, these were early days and the criteria for the neuropathologic evaluation of Alzheimer’s disease were in their infancy. For example, Fig. 3 shows excerpts of one of the first Alzheimer’s disease cases accepted into the brain bank in 1986. Gross examination revealed atrophy and enlarged ventricles. Notably, microscopic analysis was limited to sections of frontal cortex, temporal cortex, occipital cortex, hippocampus, nucleus basalis, and midbrain. Immunohistochemistry was not yet developed for diagnostic work and so diagnoses relied on histologic stains including hematoxylin and eosin, Bodian and thioflavin-S. While amyloid plaques and neurofibrillary tangles were recognized to be associated with Alzheimer’s disease dementia, John’s analysis was prescient in that he noted the presence of co-morbid Lewy bodies which at the time were of uncertain significance.

Fig. 2

Faculty of the Department of Pathology & Laboratory Medicine, University of Pennsylvania. John Q. Trojanowski is the rightmost person of the second row from the bottom sporting a bow tie and moustache. Some others in the picture include Chester Zmijewski, Carolyn (Earnst) Grotkowski, Ted Enterline, John Tomaszewski, Nick Gonatas, Harvey Kleiman, Peter Nowell, Len Jarrett, Harold Wurzel, Jack Brooks, Giuseppe Pietra, Barbara Atkinson, James Wheeler, David Goodman, David Elder, Bill Hickey, Richard Cheney, Sydney Kahn and Jerome Strauss

Fig. 3

Early Brain Bank Neuropathology Report

With the enthusiasm and vigor for which we remember John, he recruited dieners and house staff in the hospital to join him in his efforts. Residents at that time would call these cases “Troj Rockets” as they seemed to come in from nowhere but required quick work to get the tissues preserved for further scientific study. Today, this collection has grown to over 2000 cases and is known for the diversity of neurodegenerative diseases that span from normal aging to Alzheimer’s disease, frontotemporal degeneration, Parkinson’s disease and other movement disorders, and motor neuron disease. John’s talents and expertise extended well beyond the study of brain tissue. For example, he made considerable contributions to the field of fluid biomarkers as the Biomarker Core leader of the Alzheimer’s Disease Neuroimaging Initiative with his colleague Les Shaw. However, I concentrate here on his immense contributions to and impact on the field of neuropathology. The discoveries that have come from the study of these brain tissues are remarkable and profound, reflecting John’s lifelong ambition and dedication to understanding aging-related neurodegenerative diseases.

Champion of neuropathology

There has been a long-standing debate in academic medicine over the role of pathology in driving our understanding of human disease. The anatomic basis of disease was first considered by Giovanni Morgagni, the “father” of anatomic pathology, who published On the seats and causes of disease as investigated by anatomy in 1761 describing a clinicopathologic series of ~ 700 cases [15]. He suggested that diseases are not distributed throughout the body, as theorized earlier by Aelius Galenus who suggested that disturbances of the four humors were the cause of human disease, but rather that disease originates locally in specific organs and tissues. For example, Morgagni wrote about “An elderly woman whose limbs had been ulcerated for a long time, but which had gradually ceased to discharge, [who] progressively sunk to her grave as if under the pressure of age … The lateral ventricles of the brain were full of turbid serum…” [15]. Perhaps this may be one of the earliest descriptions of hydrocephalus ex-vacuo.

Over the next century, the clinicopathologic correlation study became a cornerstone of biomedical research, underpinning our understanding of human disease, amplified by the transition towards microscopic pathology by Rudolf Virchow who argued that there was a cellular basis for human disease. Indeed, the founders of the American Association of Physicians (AAP), a venerable honor society for physician-scientists which was founded in 1886 and still exists today, considered naming the fledgling organization the “Association of Physicians and Pathologists” to reflect the strength afforded to the clinicopathologic correlation study. However, at the turn of the twentieth century, American academic medicine was at a turning point where allopathic medicine was competing with homeopathic, chiropractic, and osteopathic medicine for dominance. To find a way forward and with the rise of experimental techniques that emphasized physiology, it was thought that the “clinicopathological correlation was reaching the limits of its explanatory possibilities. As the twentieth century dawned, AAP members by and large remained solidly rooted in the ideas and ideology of the previous century” [6]. Younger American academic physician-scientists believed “that contemporary clinical research should not be founded on the “dead house” science of pathology that had characterized the previous generation, but on the active, progressive science of physiology” [6]. These sentiments gave way to the founding of the American Society for Clinical Investigation, another honor society for young physician-scientists, where the “goal was to move clinical research away from what they saw as a stagnating emphasis on pathological correlation and toward a scientific basis grounded in new, more progressive sciences” [6]. While there will continue to be differing opinions on the matter, it is important for pathologists to realize that there is an undercurrent of thought where some consider pathology to be a correlative, descriptive science of limited utility.

Understanding these biases and opinions regarding the relevance of pathology in biomedical research, John was a champion of neuropathology. He firmly believed that studying human tissue was a solid basis for understanding human disease, a way to ensure that his scientific journey would always remain true to the human disease and not an artifact of an artificial experimental model. However, what was needed were modern methods to study tissue, to better glean basic and foundational insight into disease. In a remarkable boon to us all, John found a partner to join him in this pursuit.

John did an internship in internal medicine before his clinical training in pathology at Massachusetts General Hospital. At a local Boston bar, John met a postdoctoral fellow from Harvard Medical School named Virginia M.-Y. Lee who turned out to have remarkable talents in biochemistry. After Virginia moved to Philadelphia in 1979 to join what was then known as Smith-Kline & French (now GlaxoSmithKline) as an Associate Senior Research Investigator, John followed in 1980 to finish his neuropathology training at the Hospital of the University of Pennsylvania. They both became faculty at the University of Pennsylvania in the Department of Pathology and Laboratory Medicine in 1981. John and Virginia combined their talents and ambitions, eventually turning to their efforts to understand aging related neurodegenerative diseases. This marriage of neuropathology and biochemistry proved to be remarkably powerful.

By 1991, there was growing evidence that tau proteins were found in the paired helical filaments (PHFs) in Alzheimer’s disease neurofibrillary tangles. Original reports that neurofilament proteins helped form paired helical filaments were thought to be erroneous due to antibody cross-reactivity. Other antibody-based methods added weight to the presence of tau protein in PHFs. However, there was still uncertainty about the relative composition of tau versus other proteins in these enigmatic structures. For example, a review published in Neuron in April of 1991 stated that “since the quantitative depolymerization of PHFs has not been reported, one cannot yet be certain whether proteins besides tau are intrinsic constituents of these abnormal fibers” [21]. Likely due to the time it takes to publish a finished manuscript at the time, this review went to press without the inclusion of the definitive study by John Trojanowski and Virginia Lee published in Science just 2 months earlier [12]. Using tissues from John’s brain bank collection, paired helical filaments from AD brains were purified to homogeneity. Biochemical analysis of these pure PHF filaments demonstrate once and for all that PHFs were comprised solely of phosphorylated tau protein, effectively ending any controversy about the molecular composition of neurofibrillary tangles.

The aforementioned cross-reactivity of anti-neurofilament antibodies was also used at that time to help diagnose Lewy body diseases as they sometimes cross reacted with Lewy bodies. However, the biochemical composition of Lewy bodies was not known. In 1997, a mutation in SNCA was found to cause familial Parkinson’s disease in four kindred [18]. Quickly, Maria Spillantini with her partner Michel Goedert tested whether their α-synuclein antibodies labeled Lewy bodies, but they needed additional brain tissues to more comprehensively determine whether α-synuclein was the protein that forms Lewy bodies. Again, tissues from John’s brain bank collection were instrumental in demonstrating for the first time that α-synuclein is the major constituent of Lewy bodies in Parkinson’s disease and dementia with Lewy bodies [22].

Finally, there was a growing recognition that there was another protein culprit that was yet unidentified. Anti-ubiquitin antibodies had demonstrated that protein inclusions in cases clinically diagnosed with frontotemporal dementia, a neuropathologic entity called at that time frontotemporal lobar degeneration with ubiquitin positive (and tau/α-synuclein negative) inclusions, or FTLD-U. Again, John and Virginia rolled up their sleeves to identify this mystery protein. By then, their expertise had grown to include the making of specific and sensitive antibodies. Knowing that proteinaceous inclusions are typically biochemically insoluble, brain tissues from John’s brain bank collection were biochemically fractionated and the insoluble fractions were used as immunogens to create monoclonal antibodies that recognize the pathologic inclusions in FTLD-U. Using these antibodies and mass spectrometry, they identified TDP-43 protein as the major constituent of these mystery inclusions. Given that a significant proportion of individuals with amyotrophic lateral sclerosis develop frontotemporal dementia, they also demonstrated that TDP-43 protein is the major constituent of inclusions in most forms of amyotrophic lateral sclerosis [16]. Since this discovery, there have been nearly 3500 publications related to TDP-43 from almost 12,000 unique authors of which John’ centrality is indisputable (Fig. 4).

Fig. 4

Network analysis of the 11,467 authors who have published on TDP-43. Each author is designated a red node, connected to coauthors by lines. John Q. Trojanowski is shown in yellow. Courtesy of Corey T. McMillan

Here, I note that the biases against “observational” or “descriptive” pathology studies that arose in the early 1900’s are not confined to history. At the time that John and colleagues discovered TDP-43 in FTD and ALS, experimental work in ALS was dominated by studies on mutant SOD1. Justifiably, SOD1 mutations were known to cause human ALS, and the mutant SOD1 mouse exhibited motor neuron disease that was remarkably similar to the human condition. The SOD1 mouse model represented a watershed moment where the field recognized that human neurodegenerative disease can be modeled in transgenic mice. However, in humans, TDP-43 inclusions were found in nearly all forms of ALS except for those cases with SOD1 mutations [14]. The implications were clear where ALS due to mutant SOD1 appeared to represent a distinct subtype of ALS, raising questions as to whether experimental studies based on mutant SOD1 were relevant to all other forms of ALS.

Counter to this argument were questions such as “could [TDP-43 inclusions] represent merely superfluous junk?” and comments such as “Well, perhaps the “patho”—is correct, but to date, there is no “physiology” to substantia this claim. Thus reasonable/rational scientific interpretation would limit the biology of these TDP-43 aggregates—to a purely descriptive category. Until substantially more is known about the biology of the protein and the real physiology is achieved, they will remain a microscopic curiosity, but perhaps one worth watching closely” [20]. The same arguments that denigrate pathology as a science persist. However, in science as in life, being correct is also important. Together, John and Virginia argued that tau, α-synuclein, and TDP-43 are key to understanding the mechanisms that cause aging related neurodegeneration. These are the three henchmen that characterize the vast majority of all aging related neurodegenerative disease proteinopathies. The scientific impact of these discoveries is profound. We should also recognize the effect that these studies have in championing pathology as a discipline through which we can understand the human condition.

With these major accomplishments, John’s insight into disease was always colored by his experiences and expertise as a neuropathologist. Thus, while the field was squarely focused on beta-amyloid as the major driver of neurodegeneration in Alzheimer’s disease, John drew attention to tauopathy as an important but understudied pathologic substrate for dementia. Indeed, together with Brad Hyman, John led the formation of the NIA-Reagan neuropathologic criteria for the diagnosis of Alzheimer’s disease which added the requirement to ascertain the amount and extent of neurofibrillary degeneration [7]. Importantly, John did not say that beta-amyloid was not important. In fact, as a graduate student, I and several others actively worked with John and Virginia on beta-amyloid and APP processing in cellular and animal models. Rather, John argued that there was more than beta-amyloid contributing to neurodegeneration in humans.

Later, with the discovery of α-synuclein in Lewy body diseases, John together with colleagues such as Howard Hurtig would emphasize that Parkinson’s disease and related disorders is not confined to substantia nigra degeneration but affects the neuraxis more broadly, that cognitive dysfunction was an important feature of Parkinson’s disease, and that α-synuclein is an important driver of cognitive dysfunction not only in Lewy body disease but Alzheimer’s disease and aging. His arguments predated current concepts showing that Lewy body disease does not begin in the substantia nigra but rather deeper in the brainstem, sometimes associated with prodromal REM sleep disorder, and later associated with spread into the cerebrum causing dementia. The argument continued where John argued that TDP-43 proteinopathy also drives dementia not only in FTLD but also in aging and Alzheimer’s disease, now codified as the entity called Limbic-predominant Age-related TDP-43 Encephalopathy (LATE).

The importance of understanding these complex interactions was so important to John that we have worked over the last several years to bring the analysis of his entire brain bank up to modern standards. Indeed, since the founding of the brain bank, the neuropathologic criteria for Alzheimer’s disease has evolved from the Khachaturian criteria (1984) to CERAD (1991), NIA-Reagan (1997) and the current NIA-AA criteria (2012). Similarly, α-synuclein or TDP-43 inclusions could not be adequately assessed prior to his seminal discoveries. Thus, we have arduously backfilled our cases, including the 1986 “Alzheimer’s disease” case highlighted above (Fig. 3) where reassessing the case has revealed that Lewy bodies and LATE were additional contributors to neurodegeneration. John observed from the very beginning that neurodegeneration in human brains is heterogeneous and complex, and he consistently reminded us that we need to consider comorbid neuropathologies to fully understand human neurodegenerative disease [19].

Finally, over the last decade, John and Virginia have been focused on the cell-to-cell transmission of neurodegenerative disease pathologies. The studies of Braak showing a stereotyped progression of neurofibrillary degeneration in aging brains are important in that it supports a model where tauopathy spreads through the neuronal connectome. As a neuropathologist, knowing that neuropathology studies supported his hypothesis was vital to his decision to pursue this topic. This had led to highly influential studies showing that inoculation of various experimental models with brain derived pathologic proteins drives proteinopathy [13, 17]. Again, many of these studies often relied on John’s brain bank as a source of proteopathic seeds. These studies also inspired John to stage other neurodegenerative diseases to better understand how diverse diseases spread through the human brain and spinal cord including ALS, FTLD-TDP, PSP, Pick’s disease, MSA, ARTAG, and LATE [1,2,3,4,5, 8,9,10,11, 24]. The breadth and depth of John’s contributions to the scientific literature is remarkable.

These represent just a sampling of John’s over 1400 publications. Notably, over 100 of these publications were in Acta Neuropathologica, the earliest publication being from 1983 describing the expression of neurofilament proteins in various tumors using novel monoclonal anti-neurofilament antibodies [23]. John was a strong advocate for this journal, working with Werner Paulus, prior editor-in-chief, and with Johannes Attems, current editor-in-chief, to help grow and maintain the high impact of Acta Neuropathologica.

John’s long shadow

John was born in 1946 and was the second eldest of seven children. His stern father was an officer in the United States Air Force which meant John spent his childhood moving from place to place across the United States and abroad, perhaps contributing to his later penchant for experiencing the world. He would later confide in me that he chose not to have children of his own because his helping take care of his younger siblings already fulfilled his parenting instincts. This belies the fact that John nurtured the careers of countless neuropathologists and scientists. The warmth of his fatherly mentorship was a stark contrast to his strict military upbringing. His neuropathology family pedigree reaches across the globe. I evidenced this personally as condolences rolled in like waves when people learned of his untimely death with the rising of the sun across the Americas, Europe and Asia. John’s leadership was exemplary as the William Measey—Truman G. Schnabel, Jr., M.D. Professor of Geriatric Medicine and Gerontology endowed chair, Director of the Penn Institute on Aging, Director of the Penn Alzheimer’s Disease Core Center, Co-Director of the Center for Neurodegenerative Disease Research, Director of the Penn Udall Center of Excellence for Parkinson’s Disease Research, and Director of the Penn U19 Center on Alpha-Synuclein Strains in Alzheimer Disease and Related Dementias. Despite all these commitments, John was ever present in our lives as a mentor, colleague and friend. Thus, we endow upon his legacy not only his profound contributions to our understanding of neurodegenerative disease, but the legions of neuropathologists and scientists that he mentored for over decades of research.

On a personal note, I have spent nearly my entire academic career at the University of Pennsylvania, first moving here in 1997 for medical and graduate school (Fig. 5) and remaining throughout my clinical training, postdoctoral fellowship and faculty appointment. A tremendously important factor in staying here is the tremendous support of my mentors, most notably John. However, while there are benefits for staying at your home institution, when transitioning to a tenure track faculty position, there is a certain pressure to demonstrate independence from your mentors. The ability to stake out one’s own path is, for better or worse, a criterion for promotion. Thus, I found myself being drawn to John’s vibrance and enthusiasm while at the same time attempting to grow out from under his shadow. John was indeed a physically tall man at six feet four inches, but his academic stature was even taller. With his passing, I find myself yearning for his shadow which I know now is not dark, but rather cast by the tremendous light that fueled his passion for science and life.

Fig. 5

Virginia M.-Y. Lee and John Q. Trojanowski celebrating Edward B. Lee’s dissertation defense