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Alzheimer’s disease (AD) is a chronic disease characterized by a progressive decline in memory and cognition. AD progression is closely correlated with neuropathological changes and accumulation of the two main hallmark lesions, senile plaques (SP) and neurofibrillary tangles (NFT). Nevertheless, deciphering the complex biological aspects of AD requires not only looking for the neuropathological changes as the cause but rather as the collective responses to a disease process that are essential to maintain life during aging but ultimately generate a non-functional brain. Chronic conditions, such as AD, represent a new homeostatic balance or disease state, where the organism responds or adapts to maintain life. The pathologic diagnosis of AD still remains the gold standard for precise diagnosis of dementia, commonly in conjunction with cognitive-memory tests and brain image scans. Here, we present a general overview of the main neuropathological hallmarks and features of AD and related dementia, revealing the key biological and functional changes as potential drives of age-dependent brain failure related to AD. The present work reflects some of the main ideas presented during the ASIP Rous-Whipple Award Lecture 2021.
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by memory loss, diminishing mental functions, and cognitive impairment.
The article describes an unknown illness characterized by a rapid loss of memory, disorientation, altered behavior, marked difficulties in reading, misspelling, and disorientation, that progressed in severity over 4.5 years of observation. A post-mortem examination showed an evenly atrophic brain and arteriosclerotic changes. Brain tissue revealed striking changes in the neurofibrils, and the presence of thick fibrils in the neurons. He observed that “the change of the fibrils seems to be a parallel process of deposition of a pathological metabolic substance in the neurons whose closer examination is still pending.” Additional interesting comments from Alois Alzheimer included the observation that around 1/4 to 1/3 of the neurons in the cortex had morphological changes, and also marked neuronal loss in the upper layers of the brain cortex. He concluded that “we are dealing with a special illness” and that “a histological examination will enable us to determine the characteristics of some of these cases.” It is now recognized that elderly patients with AD and related dementia have an abnormal aggregation of misfolded proteins forming neurotoxic bundles within the cerebral cortex.
Neuropathological hallmarks of Alzheimer’s disease
The hallmark pathologies of AD are the accumulation of amyloid-β (Aβ) peptide into senile plaques (SP) outside neurons and of twisted strands of hyperphosphorylated tau protein into neurofibrillary tangles (NFT) inside neurons in the brain.
Figure 1 shows immunocytochemistry location of SP and NFT in AD brain tissue. The deposition of these protein aggregates is accompanied by complex molecular and cellular responses that lead to synaptic failure, neuroinflammation, and eventually progressive neuronal death.
Over the last decades, one of the main focuses in AD has been to decipher the mechanisms of formation of SP and NFT, with the objective of finding potential avenues to stop or even reverse their formation. This knowledge could be used to reduce the development of AD and also for the development of a possible cure or effective treatments of AD and related dementias.
The causes of AD are not completely understood but probably include a combination of many factors, including aging, genetics, environmental conditions, and lifestyle. Even though aging is the most important risk factor for neurodegeneration, it is not a direct cause of AD, only one-third of all people age 85 and older may have AD, and many elderly never develop dementia (www.nia.nih.gov/health/what-causes-alzheimers-disease, Access 06/07/2022). Findings of SP and NFT in normal aging and in patients with traumatic brain injury are common, although neither fully correlated with cognitive loss, but they were seldom questioned as to the unique cause of AD.
AD is classified into two types, early-onset familial AD (eFAD) and late-onset or sporadic AD. Remarkably, late-onset or sporadic AD is by far the most common type of AD, whereas only 1% or less of all cases corresponds to eFAD. Genetic factors are implicated in both types of AD. In particular, the apolipoprotein E-ε4 (APOE-ε4) gene on chromosome 19 has showed a higher risk of AD, but inheriting an APOE-ε4 allele does not definitely correlate with the development of AD since many carriers simply do not develop dementia. Moreover, APOE-ε3 gene is the most common type, with no evidence suggesting it may decrease or increase the risk of AD. In contrast, APOE-ε2 is a rare form of this gene that may provide some protection or lower risk against AD. Subjects that inherit genes with specific mutations associated with eFAD usually develop symptoms as early as their late 30s and mid-60s. In particular, genetic studies of AD subjects have identified several mutations in three genes: amyloid-β precursor protein (AβPP) on chromosome 21, presenilin 1 (PSEN1) on chromosome 14, and presenilin 2 (PSEN2) on chromosome 1. The function of AβPP is not completely clear, but probably implicated as a neuronal receptor, in synaptic formation, and also on hormones-metabolites regulation. AβPP is the precursor of Aβ peptide, which is produced by its proteolytic processing by PSEN1, PSEN2 and other enzymes. Identification of mutations in AβPP, PSEN1, and PSEN2 genes were associated with eFAD and an overproduction of Aβ peptide that forms neurotoxic fibrils and SP. Nevertheless, the association of these genes with AD has been misinterpreted or placed as directly causal. Causality requires demonstrating that Aβ removal either prevents or reverses the development of AD.
Unfortunately, there are no effective treatments for AD. Current treatments only focus on the management of symptoms and maintenance of mental function, with limited effectiveness. Over the last years, the main approach for the development of therapeutic compounds against AD has been focused mainly on Aβ. In particular, with the development of multiple monoclonal therapeutic antibodies intended for the removal of neurotoxic forms of Aβ, but surprisingly, in over a dozen clinical trials, the AD patients have not benefited significatively, as expected or as causality would dictate.
The cumulative research indicate that Aβ is not the only driving force of AD, but still raises the question of what Aβ is doing that genetically and biologically associates it to the development of AD pathology causing progressive neuronal failure. In the same direction, therapeutic antibodies targeting aggregated tau into NFT are being evaluated for removing tau deposits from the brain, but initial studies focused on tau removal have also not reversed disease progression. The failure of multiple therapeutic compounds (small drugs, inhibitors, antibodies) targeting Aβ, and tau open the question of whether the biological roles of Aβ and tau in AD are still incomplete, wrong, or even completely reversed. There is a striking mismatch between lesions of SP/NFT and symptoms of AD; a significant number of subjects show neuropathological changes with widespread deposition of SP/NFT (observed at autopsy) but with relatively normal cognitive conditions and without manifesting dementia.
As discussed previously, the etiology and pathogenesis of AD are not completely described, but oxidative stress is a key component. Oxidative stress refers to a state of cellular imbalance between the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) with the antioxidant defense systems.
Proteins, lipids, and genetic material may suffer oxidative damage during oxidative stress, altering their functions-structure and promoting mutations. In the AD brain, the amount of oxidative damage is increased in comparison with healthy elderly individuals. Including elevated levels of intracellular ROS/RNS, protein carbonyls and 3-nitrotyrosine, increased lipid peroxidation, significant DNA damage as 8-hydroxy-deoxyguanosine (8OHdG) produced by ROS/RNS, and 8-hydroxyguanine (8OHG) in RNA.
Almost three decades ago, we performed pioneering studies of oxidative damage in AD, particularly to understand the chemical and biological properties of SP and NFT. In brain tissue from AD, we identified oxidative modification of neuronal proteins through nitration, and these changes were also located within NFT (Figure 2 A-B).
Conversely, the levels of nitro-tyrosine in age-matched controls were undetectable through immunocytochemistry in the cerebral cortex. Furthermore, oxidative damage in AD is also present in lipid membranes, as demonstrated by immunocytochemical detection of lipid peroxidation (4-hydroxynonenal) in brain tissue from AD patients (Figure 2 C-D).
Surprisingly, the lipid peroxidation collocated with NFT lesions but not with SP. The oxidative damage in AD is extended to the genetic material, as demonstrated by identifying 8OHdG and 8OHG in neurons within the hippocampus, subiculum, entorhinal cortex, as well as frontal, temporal, and occipital neocortex (Figure 2 E-F).
The subcellular localization of the oxidative damage mainly in the cytoplasm allowed us to hypothesize that mitochondrial components may be the source of free radicals promoting oxidative stress in AD. The signs of oxidative damage are more prominent early in the disease and reduces with disease progression, this relationship is more significant in ApoE-ε4 carries.
With these studies, we concluded that the damage of oxidative stress conditions is widely extended in AD to all biomolecules, including lipids, proteins, sugars, and nucleic acids in brain cells. Moreover, the oxidative damage to each type of biomolecule specifically increased in vulnerable populations of neurons during AD. The range of oxidative damage types in AD suggested the involvement of Fenton reactions as the source of free radicals that promote oxidative damage, particularly focusing on abnormal levels of redox-active metal ions, such as copper and iron, which are the source of ROS and other redox-generated free radicals.
Mainly by affecting the hippocampus and temporal cortex with oxidative damage, and mostly restricted to the neuronal components in the cytoplasm, not APC and NFT. In fact, affected neurons with NFT had reduced oxidative damage, and Aβ levels negatively correlated with oxidative damage in AD and Down syndrome.
Overall, these observations suggested that the overexpression and aggregation of Aβ and tau could be part of complex neuronal molecular responses that are responsive to oxidative stress conditions in the aging brain. To gain further insight, we performed experiments to understand the brain responses to oxidative stress. First, we determined that NFT are not formed by the aggregation of pure tau protein but also are intimately associated with other proteins, such as heme oxygenase-1, an enzyme that converts heme to biliverdin/bilirubin—transforming an oxidant to an antioxidant.
Second, the levels of tau phosphorylation are correlated with heme oxygenase-1 expression in neuronal cells, and that tau is regulated through signal transduction pathways that are modulated by oxidative stress to play a role in the cytoprotection of vulnerable neurons.
The interactions between Aβ peptide and redox-active metals ions promoted the oxidation of His residues of Aβ, and favored its aggregation into APC.
In recent studies we demonstrated APC from AD contain metallic elements within them, including copper and iron in multiple valence states (Cu+, Cu2+, Fe2+, and Fe3+), as observed with synchrotron-based X-ray spectromicroscopy (STXM) (Figure 3 C-G).
The advanced imaging and quantitative spectroscopic techniques allowed to directly demonstrate the involvement of redox-active metal ions in redox cycling metabolism. Furthermore, advanced STXM imaging revealed the presence of nanometer size metallic aggregates of copper (Cuᵒ) and iron (Feᵒ) within purified amyloid plaque cores, and in correlation with deposition of iron in AD pathology. Remarkably, APC from advanced AD also contained iron aggregates formed by redox-active species, Fe+3, Fe+2, and metallic iron (Feᵒ) and forming nanostructured aggregates.
Some of these metallic species should not be stable to air, demonstrating Aβ peptide has unique properties responsible for its antioxidant activity, this through binging of redox-active ions and promoting their aggregation within APC.
Overall, it was observed not only do SP and NFT play a role in oxidative stress responses in brain cells, but that several signal transduction pathways are induced during the development of neurodegenerative processes of AD, such as mitogen-activated protein kinase/extracellular receptor kinase (MAPK/ERK),
Mitochondrial dysfunction in the pathogenesis of Alzheimer’s disease
Another neuropathological feature related to the neurodegenerative processes of AD is mitochondrial dysfunction. Specifically, mitochondrial structural and functional integrity alterations, mitochondrial biogenesis and dynamics, axonal transport, mitophagy, and mitochondrial proteostasis.
These biological changes raise the question of where and how they occur in vulnerable cell populations in the AD brain. We focused our efforts on elucidating the mitochondrial responses during AD, because mitochondria contain abundant levels of metalloproteins and are the main source of free radicals and highly oxidant compounds such as superoxide (O2-). Just as the majority of oxidative damage is dependent on redox-active ions Cu/Fe, the mitochondria oxidative metabolism is almost completely dependent on these metals as cofactors and acceptors. Mitochondria from AD brain samples were examined with probes to mitochondrial DNA (mtDNA), enzyme proteins, and enzyme-prosthetic groups, such as cytochrome oxidase 1. We found that the same population of neurons displaying a high degree of oxidative damage also contained abundant mitochondrial debris, mostly located within autophagosomes.
Close examination of an aging brain series revealed those after age 40 or older showed similar—although reduced—mitochondria autophagy to that observed in patients with AD. These observations suggested a new molecular mechanism related to AD pathogenesis, one where the activation of mitochondria autophagy is at the center of neuronal damage. Figure 4 illustrates the neuronal responses to oxidative stress and its impact on mitochondrial dysfunction. Our studies of mitochondria transport, fusion-fission, and turnover all have consistently shown morphological and functional abnormalities related to the progression of AD. Activation of mitochondria autophagy poses several challenges for affected neurons. Specifically, the alterations in metal ion transport/homeostasis are key components, since redox-active Cu/Fe ions that are liberated during the process of mitochondria turnover can cause massive oxidative damage to all compartments of neurons and even the entire brain if not properly counterbalanced by the antioxidant systems. From the cumulative evidence, we hypothesize that Aβ and tau may play this role as a neuroprotectant by binding free metal ions to reduce their reactivity. This molecular function of Aβ and tau is essential during neurodegeneration, and mutations in AβPP may lead to improper functionality in neuronal cells and, consequently, lead to early-onset AD.
Mitochondria are at the center of aerobic energy production, producing over 95% of all cellular ATP, also controlling programmed cell death processes, and a master neuronal signaling, calcium (Ca2+). Calcium controls microtubule assembly, and these microtubules are decreased by up to 90% in neurons affected by AD, as well as diminished in the brain during normal aging.
Proteolysis is also controlled by calcium, as is synaptic vesicle fusion. Loss of mitochondrial functions seems to be at the center of oxidative stress responses, promoting autophagy, altered redox-active biometal dynamics, and activation of neuronal survival mechanisms in neurodegeneration. During aging, diminished mitochondrial functions are met with critical compensations by Aβ/tau/HO-1/autophagy/survival responses that are all essential to maintaining brain function. As aging progresses, the compensation mechanisms deployed in the brain grow and may ultimately reveal themselves as “pathology.” With continued aging, some of the compensation mechanisms could fail to maintain normal brain function and rather redirect cellular metabolism to survival rather than function. This choice is evolutionarily selected as a response to temporary brain stress insults rather than the ever-present effects of normal aging. This interpretation is consistent with the lack of correlation between neuronal loss and cognition: simply, in AD, the neurons are there but not functioning.
While these affected neurons might correspond to senescent cells, all neurons of the same population display pre-apoptotic changes without death, and their therapeutic removal will only advance disease progression. Moreover, mutations in AβPP lead to the improper molecular processing of Aβ rather than primary causation. Finally, and most significantly, the removal of Aβ, tau, or even redox-active metals will not benefit AD patients by reversal of the primary driver because each is an important neuroprotective response, explaining why many therapies focused on Aβ removal have had no benefit in cognition, memory, and brain function.
Mitochondria are highly responsive to changes in cell homeostasis, cellular stress, inflammation responses, environment, nutrition, and exercise status, all of which play major factors in the potential development of AD. All these factors are directly or indirectly linked to the dysfunctions in energy metabolism as key drivers to the development of AD. Potential therapies maintaining mitochondrial functions will delay AD, as observed with some anti-diabetes drugs (metformin, insulin, among others). Similarly, promoting more efficient autophagy or restoring calcium levels could lower neuronal oxidative stress. Focusing on the complex biology of AD through pathology will continue to reveal potential or alternative therapeutic insights that will benefit AD patients and their families.
Alzheimer’s disease is still a complex chronic neurological disease with no effective treatments. The molecular and structural changes observed in AD brain have helped to establish the key mechanisms involved in the progression of the disease, and to differentiate them from normal aging processes. The main neuropathological features are synaptic failure, neuronal loss, and mainly the widespread presence of amyloid plaque cores and neurofibrillary tangles, but these abnormal protein aggregates are more complex than we thought. Over the last years, with the advancement of novel quantitative and advanced imaging techniques, the ultrastructural and chemical signatures of SP and NFT revealed the presence of inorganic aggregates formed by biometals such as Fe, Cu, Zn and Ca. Furthermore, additional pathological features not frequently analyzed include alterations in mitochondria, lipids, inflammation responses, and altered neuronal phenotype. Remarkably, clinical decisions are often based on histopathological/microscopic results, such as in cancer, but in the case of AD this is not possible, and the confirmation of neuropathology is performed postmortem. We still face significant challenges to fight AD, expecting that soon we will have effective therapeutic approaches, and sensitive/effective molecular diagnostic tools.
Semmes Foundation, Lowe Foundation, Kleberg Foundation, Alzheimer’s Association, NIH National Institute on Aging, and UTSA Brain Health Consortium.
Funding: Semmes Foundation, Lowe Foundation, Kleberg Foundation, Alzheimer’s Association (AARFD-17-529742), NIH National Institute on Aging (R01AG066749).
Disclosures: none declared
Footnote: The Rous-Whipple Award is given by the American Society for Investigative Pathology (ASIP) to a senior pathologist with a distinguished career in experimental pathology research and continued productivity at the time of the award. George Perry, Ph.D., recipient of the 2021 ASIP Rous-Whipple Award, delivered a lecture entitled “Pathology in Alzheimer Disease: A Protective Response?” on April 27, 2021, at the 2021 ASIP Annual Meeting at Experimental Biology (held virtually).