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Apolipoprotein E protein isoforms, particularly ApoE4, appear to accelerate brain-wide tau propagation that eventually leads to neuronal injury and death in a manner independent from amyloid-beta, according to findings from transgenic mouse model studies.
“We found ApoE itself, especially ApoE4, was essential to neuronal death,” wrote first author Yang Shi of Washington University, St. Louis, and her colleagues, led by David M. Holtzman, MD, in new research published in Nature. “With pathological tau accumulation, the presence of ApoE, especially ApoE4, may make neurons more susceptible to degeneration, whereas the absence of ApoE may protect neurons from death.”
The new research also illustrates a differential effect between the three APOE alleles. In the team’s in vivo study, tau-expressing mice with the APOE4 allele were most affected, and those with the E3 and E2 versions progressively less so. Mice that didn’t express the human gene at all showed no change in tau and no immune reaction (Nature. 2017;549:523-7).
This new picture of tauopathy – a common feature in Alzheimer’s, frontotemporal dementia, corticobasal degeneration, Pick disease, and progressive supranuclear palsy – suggests an expanded role for ApoE, which until now has been associated mostly with increased amyloid deposition in the Alzheimer’s disease (AD) brain.
“I think this paper is potentially very important, identifying what appear to be strong connections between tau and ApoE that we had no idea about before,” Dr. Wolfe said in an email. “While independent confirmation is needed, this new work is coming from a strong research team that has made other seminal discoveries in the field. Uncovering the molecular basis for ApoE’s effect on tau pathology and glial cell activation may suggest new targets for drug discovery for AD.”
Evidence of ApoE4’s greater impact
To examine ApoE’s effect on tau, the research team bred new lines of genetically modified mice, all of which expressed human tau. Some also expressed human ApoE4, E3, or E2 in place of mouse ApoE. A comparator mouse expressed tau, but not ApoE.
By the time the mice were 9 months old, the tau-E4 strain showed significantly more brain atrophy than did the tau-E3 and tau-E2 strains. The mice who expressed tau but were free of ApoE showed no brain changes.
A closer look showed that atrophy occurred primarily in areas associated with the cognitive changes seen in dementia: the hippocampus, piriform/entorhinal cortex, and amygdala. The ventricles were also enlarged.
“These results revealed an important role of ApoE in regulating tau-mediated neurodegeneration, with ApoE4 causing more severe damage and the absence of ApoE being protective,” the investigators wrote.
The E4/tau tango started early, too, the team noted. At 3 months old, tau-E4 mice had no obvious brain atrophy, but already had significantly more soluble tau than did any of the other strains. By 9 months, when tauopathy was obvious, the tau-E4 mice still had more of the protein, which had shifted from a soluble to an insoluble and hyperphosphorylated state. The tau-E4 mice didn’t appear to be making more tau than the others, though; rather, they were less able to clear it through the neurons’ clearing and recycling system of autophagy.
Drilling down further into the neurons’ pathophysiology, the team found that tau first appeared in the axons of dentate gyrus granule cells in the hippocampus, and then, at an early age, moved into the cell body. Again, there were APOE allele–specific patterns to tau propagation. The team saw four major tau staining patterns, which correlated with the level of brain atrophy. Types 1 and 2, associated with least atrophy, occurred most often in the tau-only, ApoE-negative mice; type 4, associated with the greatest atrophy, occurred most often in the tau-E4 mice.
“The featured distribution of these ... patterns, which either represent different tau structures or progressively more advanced pathological tau stages, indicate ApoE affects either tau conformation or tau pathology progression,” the investigators wrote.
Greatest neurodegeneration seen with ApoE4
Tau-mediated neurodegeneration initiated levels of inflammatory response that also depended on the type of ApoE isoform. Exposure to a culture of damaged neurons and mixed glial cells caused microglia to release a flood of inflammatory cytokines that called in a host of astrocytes to kill damaged tau-E4 neurons en masse, but attacked the tau-E3, tau-E2, and tau-only strains much less. This finding indicates that “ApoE itself was directly involved in inducing neurotoxicity in tau-expressing susceptible neurons,” the team wrote.
Finally, they investigated this model of neurodegeneration in postmortem brain samples of patients with corticobasal degeneration, Pick disease, and progressive supranuclear palsy – the three most common sporadic primary tauopathies. Patients with the E4 allele showed more severe neurodegeneration and a greater interaction of tau pathology and neurodegeneration. Amyloid deposition was associated with less severe neurodegeneration.
Taken together, the findings strongly suggest that the high-risk APOE4 allele is the linchpin that links neuroinflammation to neuronal death in the setting of tau pathology, the investigators concluded.
“The presence of degenerating neurons appeared to further induce neuroinflammation, which was augmented by ApoE4 owing to its inherently higher innate immune reactivity. While activated microglia may be protective to some extent in the setting of amyloid-beta pathology, by targeting plaques and reducing dystrophic neurites, they could be deleterious in tauopathy by directly targeting injured neurons and by activating toxic astrocytes. Enhanced neuroinflammation associated with ApoE4 may further exacerbate neurodegeneration.”
The study was funded by grants awarded to multiple investigators by the National Institutes of Health, the JPB Foundation, Cure Alzheimer’s Fund, AstraZeneca, the Consortium for Frontotemporal Dementia Research, the Tau Consortium, the National Multiple Sclerosis Society, the Nancy Davis Foundation, and the Amyotrophic Lateral Sclerosis Association. Dr. Holtzman cofounded and is on the scientific advisory board of C2N Diagnostics. He consults for Genentech, AbbVie, Eli Lilly, Proclara, GlaxoSmithKline, and Denali. Dr. Holtzman’s lab is funded by institutional research grants from C2N Diagnostics, Eli Lilly, AbbVie, and Denali.
[email protected]
On Twitter @Alz_Gal
The study by Shi et al. represents an important step forward in our understanding of Alzheimer’s disease (AD) pathogenesis and raises another challenge to the amyloid hypothesis: ApoE4 enhances tau pathology independent of amyloid in vivo, in vitro and – possibly – even in humans.
The discovery that the plaques seen in AD brains were composed of amyloid-beta (Abeta) peptide, and the discovery soon thereafter that mutations of genes involved in Abeta production caused dominantly inherited AD, made clear that there was something very important about amyloid and its relationship to AD.
ApoE4 is the next most potent genetic variant to predispose to AD, and much more prevalent than the dominant mutations. When first reported in the early 1990s, the gene was something of a mystery because it did not have an apparent link to amyloid. However, research suggests that ApoE4-mediated effects may be via either amyloid-dependent or amyloid-independent mechanisms. The latter have been particularly championed by investigators at the Gladstone Institute of Neurological Disease who are working to develop a structural modifier of the ApoE4 isoform to prevent the direct toxic effects of the intraneuronal carboxyl fragment that is generated during the production of Abeta. To date, no human trials have been conducted.
Turning to the study by Shi and colleagues, in addition to their convincing mouse and in vitro data, they present data from a collection of human tauopathy brains. These show that ApoE4 was associated with greater tau pathology in patients who died with corticobasal degeneration, Pick disease, and progressive supranuclear palsy. Incidental amyloid deposition in some members of this cohort was not associated with greater tauopathy burden, in contrast to what one might have predicted if Abeta was triggering tau hyperphosphorylation. For clinicians, the human observations are particularly intriguing and deserve replication and further study.
The study of Shi et al. does not invalidate the possibility that amyloid plays a key role in AD pathogenesis. Indeed, that relationship seems firm, since the dominantly inherited AD mutations all affect Abeta production or aggregation. However, it does raise questions as to whether the dominantly inherited cases are representative of the “sporadic” and ApoE4-associated cases. It also raises questions as to the exact role amyloid plays in AD pathogenesis. Abeta toxicity has been amply demonstrated, but that does not necessarily mean that Abeta toxicity is the cause of AD or the driving force behind disease progression.
Clearly, more work is needed to clarify what role amyloid is playing in AD pathogenesis beyond existing toxicity models. Hopefully, the study by Shi et al. will stimulate new models and therapeutic ideas.
Richard J. Caselli, MD, is professor of neurology at the Mayo Clinic in Scottsdale, Ariz. He also is associate director and clinical core director of the Arizona Alzheimer’s Disease Center.
The study by Shi et al. represents an important step forward in our understanding of Alzheimer’s disease (AD) pathogenesis and raises another challenge to the amyloid hypothesis: ApoE4 enhances tau pathology independent of amyloid in vivo, in vitro and – possibly – even in humans.
The discovery that the plaques seen in AD brains were composed of amyloid-beta (Abeta) peptide, and the discovery soon thereafter that mutations of genes involved in Abeta production caused dominantly inherited AD, made clear that there was something very important about amyloid and its relationship to AD.
ApoE4 is the next most potent genetic variant to predispose to AD, and much more prevalent than the dominant mutations. When first reported in the early 1990s, the gene was something of a mystery because it did not have an apparent link to amyloid. However, research suggests that ApoE4-mediated effects may be via either amyloid-dependent or amyloid-independent mechanisms. The latter have been particularly championed by investigators at the Gladstone Institute of Neurological Disease who are working to develop a structural modifier of the ApoE4 isoform to prevent the direct toxic effects of the intraneuronal carboxyl fragment that is generated during the production of Abeta. To date, no human trials have been conducted.
Turning to the study by Shi and colleagues, in addition to their convincing mouse and in vitro data, they present data from a collection of human tauopathy brains. These show that ApoE4 was associated with greater tau pathology in patients who died with corticobasal degeneration, Pick disease, and progressive supranuclear palsy. Incidental amyloid deposition in some members of this cohort was not associated with greater tauopathy burden, in contrast to what one might have predicted if Abeta was triggering tau hyperphosphorylation. For clinicians, the human observations are particularly intriguing and deserve replication and further study.
The study of Shi et al. does not invalidate the possibility that amyloid plays a key role in AD pathogenesis. Indeed, that relationship seems firm, since the dominantly inherited AD mutations all affect Abeta production or aggregation. However, it does raise questions as to whether the dominantly inherited cases are representative of the “sporadic” and ApoE4-associated cases. It also raises questions as to the exact role amyloid plays in AD pathogenesis. Abeta toxicity has been amply demonstrated, but that does not necessarily mean that Abeta toxicity is the cause of AD or the driving force behind disease progression.
Clearly, more work is needed to clarify what role amyloid is playing in AD pathogenesis beyond existing toxicity models. Hopefully, the study by Shi et al. will stimulate new models and therapeutic ideas.
Richard J. Caselli, MD, is professor of neurology at the Mayo Clinic in Scottsdale, Ariz. He also is associate director and clinical core director of the Arizona Alzheimer’s Disease Center.
The study by Shi et al. represents an important step forward in our understanding of Alzheimer’s disease (AD) pathogenesis and raises another challenge to the amyloid hypothesis: ApoE4 enhances tau pathology independent of amyloid in vivo, in vitro and – possibly – even in humans.
The discovery that the plaques seen in AD brains were composed of amyloid-beta (Abeta) peptide, and the discovery soon thereafter that mutations of genes involved in Abeta production caused dominantly inherited AD, made clear that there was something very important about amyloid and its relationship to AD.
ApoE4 is the next most potent genetic variant to predispose to AD, and much more prevalent than the dominant mutations. When first reported in the early 1990s, the gene was something of a mystery because it did not have an apparent link to amyloid. However, research suggests that ApoE4-mediated effects may be via either amyloid-dependent or amyloid-independent mechanisms. The latter have been particularly championed by investigators at the Gladstone Institute of Neurological Disease who are working to develop a structural modifier of the ApoE4 isoform to prevent the direct toxic effects of the intraneuronal carboxyl fragment that is generated during the production of Abeta. To date, no human trials have been conducted.
Turning to the study by Shi and colleagues, in addition to their convincing mouse and in vitro data, they present data from a collection of human tauopathy brains. These show that ApoE4 was associated with greater tau pathology in patients who died with corticobasal degeneration, Pick disease, and progressive supranuclear palsy. Incidental amyloid deposition in some members of this cohort was not associated with greater tauopathy burden, in contrast to what one might have predicted if Abeta was triggering tau hyperphosphorylation. For clinicians, the human observations are particularly intriguing and deserve replication and further study.
The study of Shi et al. does not invalidate the possibility that amyloid plays a key role in AD pathogenesis. Indeed, that relationship seems firm, since the dominantly inherited AD mutations all affect Abeta production or aggregation. However, it does raise questions as to whether the dominantly inherited cases are representative of the “sporadic” and ApoE4-associated cases. It also raises questions as to the exact role amyloid plays in AD pathogenesis. Abeta toxicity has been amply demonstrated, but that does not necessarily mean that Abeta toxicity is the cause of AD or the driving force behind disease progression.
Clearly, more work is needed to clarify what role amyloid is playing in AD pathogenesis beyond existing toxicity models. Hopefully, the study by Shi et al. will stimulate new models and therapeutic ideas.
Richard J. Caselli, MD, is professor of neurology at the Mayo Clinic in Scottsdale, Ariz. He also is associate director and clinical core director of the Arizona Alzheimer’s Disease Center.
Apolipoprotein E protein isoforms, particularly ApoE4, appear to accelerate brain-wide tau propagation that eventually leads to neuronal injury and death in a manner independent from amyloid-beta, according to findings from transgenic mouse model studies.
“We found ApoE itself, especially ApoE4, was essential to neuronal death,” wrote first author Yang Shi of Washington University, St. Louis, and her colleagues, led by David M. Holtzman, MD, in new research published in Nature. “With pathological tau accumulation, the presence of ApoE, especially ApoE4, may make neurons more susceptible to degeneration, whereas the absence of ApoE may protect neurons from death.”
The new research also illustrates a differential effect between the three APOE alleles. In the team’s in vivo study, tau-expressing mice with the APOE4 allele were most affected, and those with the E3 and E2 versions progressively less so. Mice that didn’t express the human gene at all showed no change in tau and no immune reaction (Nature. 2017;549:523-7).
This new picture of tauopathy – a common feature in Alzheimer’s, frontotemporal dementia, corticobasal degeneration, Pick disease, and progressive supranuclear palsy – suggests an expanded role for ApoE, which until now has been associated mostly with increased amyloid deposition in the Alzheimer’s disease (AD) brain.
“I think this paper is potentially very important, identifying what appear to be strong connections between tau and ApoE that we had no idea about before,” Dr. Wolfe said in an email. “While independent confirmation is needed, this new work is coming from a strong research team that has made other seminal discoveries in the field. Uncovering the molecular basis for ApoE’s effect on tau pathology and glial cell activation may suggest new targets for drug discovery for AD.”
Evidence of ApoE4’s greater impact
To examine ApoE’s effect on tau, the research team bred new lines of genetically modified mice, all of which expressed human tau. Some also expressed human ApoE4, E3, or E2 in place of mouse ApoE. A comparator mouse expressed tau, but not ApoE.
By the time the mice were 9 months old, the tau-E4 strain showed significantly more brain atrophy than did the tau-E3 and tau-E2 strains. The mice who expressed tau but were free of ApoE showed no brain changes.
A closer look showed that atrophy occurred primarily in areas associated with the cognitive changes seen in dementia: the hippocampus, piriform/entorhinal cortex, and amygdala. The ventricles were also enlarged.
“These results revealed an important role of ApoE in regulating tau-mediated neurodegeneration, with ApoE4 causing more severe damage and the absence of ApoE being protective,” the investigators wrote.
The E4/tau tango started early, too, the team noted. At 3 months old, tau-E4 mice had no obvious brain atrophy, but already had significantly more soluble tau than did any of the other strains. By 9 months, when tauopathy was obvious, the tau-E4 mice still had more of the protein, which had shifted from a soluble to an insoluble and hyperphosphorylated state. The tau-E4 mice didn’t appear to be making more tau than the others, though; rather, they were less able to clear it through the neurons’ clearing and recycling system of autophagy.
Drilling down further into the neurons’ pathophysiology, the team found that tau first appeared in the axons of dentate gyrus granule cells in the hippocampus, and then, at an early age, moved into the cell body. Again, there were APOE allele–specific patterns to tau propagation. The team saw four major tau staining patterns, which correlated with the level of brain atrophy. Types 1 and 2, associated with least atrophy, occurred most often in the tau-only, ApoE-negative mice; type 4, associated with the greatest atrophy, occurred most often in the tau-E4 mice.
“The featured distribution of these ... patterns, which either represent different tau structures or progressively more advanced pathological tau stages, indicate ApoE affects either tau conformation or tau pathology progression,” the investigators wrote.
Greatest neurodegeneration seen with ApoE4
Tau-mediated neurodegeneration initiated levels of inflammatory response that also depended on the type of ApoE isoform. Exposure to a culture of damaged neurons and mixed glial cells caused microglia to release a flood of inflammatory cytokines that called in a host of astrocytes to kill damaged tau-E4 neurons en masse, but attacked the tau-E3, tau-E2, and tau-only strains much less. This finding indicates that “ApoE itself was directly involved in inducing neurotoxicity in tau-expressing susceptible neurons,” the team wrote.
Finally, they investigated this model of neurodegeneration in postmortem brain samples of patients with corticobasal degeneration, Pick disease, and progressive supranuclear palsy – the three most common sporadic primary tauopathies. Patients with the E4 allele showed more severe neurodegeneration and a greater interaction of tau pathology and neurodegeneration. Amyloid deposition was associated with less severe neurodegeneration.
Taken together, the findings strongly suggest that the high-risk APOE4 allele is the linchpin that links neuroinflammation to neuronal death in the setting of tau pathology, the investigators concluded.
“The presence of degenerating neurons appeared to further induce neuroinflammation, which was augmented by ApoE4 owing to its inherently higher innate immune reactivity. While activated microglia may be protective to some extent in the setting of amyloid-beta pathology, by targeting plaques and reducing dystrophic neurites, they could be deleterious in tauopathy by directly targeting injured neurons and by activating toxic astrocytes. Enhanced neuroinflammation associated with ApoE4 may further exacerbate neurodegeneration.”
The study was funded by grants awarded to multiple investigators by the National Institutes of Health, the JPB Foundation, Cure Alzheimer’s Fund, AstraZeneca, the Consortium for Frontotemporal Dementia Research, the Tau Consortium, the National Multiple Sclerosis Society, the Nancy Davis Foundation, and the Amyotrophic Lateral Sclerosis Association. Dr. Holtzman cofounded and is on the scientific advisory board of C2N Diagnostics. He consults for Genentech, AbbVie, Eli Lilly, Proclara, GlaxoSmithKline, and Denali. Dr. Holtzman’s lab is funded by institutional research grants from C2N Diagnostics, Eli Lilly, AbbVie, and Denali.
[email protected]
On Twitter @Alz_Gal
Apolipoprotein E protein isoforms, particularly ApoE4, appear to accelerate brain-wide tau propagation that eventually leads to neuronal injury and death in a manner independent from amyloid-beta, according to findings from transgenic mouse model studies.
“We found ApoE itself, especially ApoE4, was essential to neuronal death,” wrote first author Yang Shi of Washington University, St. Louis, and her colleagues, led by David M. Holtzman, MD, in new research published in Nature. “With pathological tau accumulation, the presence of ApoE, especially ApoE4, may make neurons more susceptible to degeneration, whereas the absence of ApoE may protect neurons from death.”
The new research also illustrates a differential effect between the three APOE alleles. In the team’s in vivo study, tau-expressing mice with the APOE4 allele were most affected, and those with the E3 and E2 versions progressively less so. Mice that didn’t express the human gene at all showed no change in tau and no immune reaction (Nature. 2017;549:523-7).
This new picture of tauopathy – a common feature in Alzheimer’s, frontotemporal dementia, corticobasal degeneration, Pick disease, and progressive supranuclear palsy – suggests an expanded role for ApoE, which until now has been associated mostly with increased amyloid deposition in the Alzheimer’s disease (AD) brain.
“I think this paper is potentially very important, identifying what appear to be strong connections between tau and ApoE that we had no idea about before,” Dr. Wolfe said in an email. “While independent confirmation is needed, this new work is coming from a strong research team that has made other seminal discoveries in the field. Uncovering the molecular basis for ApoE’s effect on tau pathology and glial cell activation may suggest new targets for drug discovery for AD.”
Evidence of ApoE4’s greater impact
To examine ApoE’s effect on tau, the research team bred new lines of genetically modified mice, all of which expressed human tau. Some also expressed human ApoE4, E3, or E2 in place of mouse ApoE. A comparator mouse expressed tau, but not ApoE.
By the time the mice were 9 months old, the tau-E4 strain showed significantly more brain atrophy than did the tau-E3 and tau-E2 strains. The mice who expressed tau but were free of ApoE showed no brain changes.
A closer look showed that atrophy occurred primarily in areas associated with the cognitive changes seen in dementia: the hippocampus, piriform/entorhinal cortex, and amygdala. The ventricles were also enlarged.
“These results revealed an important role of ApoE in regulating tau-mediated neurodegeneration, with ApoE4 causing more severe damage and the absence of ApoE being protective,” the investigators wrote.
The E4/tau tango started early, too, the team noted. At 3 months old, tau-E4 mice had no obvious brain atrophy, but already had significantly more soluble tau than did any of the other strains. By 9 months, when tauopathy was obvious, the tau-E4 mice still had more of the protein, which had shifted from a soluble to an insoluble and hyperphosphorylated state. The tau-E4 mice didn’t appear to be making more tau than the others, though; rather, they were less able to clear it through the neurons’ clearing and recycling system of autophagy.
Drilling down further into the neurons’ pathophysiology, the team found that tau first appeared in the axons of dentate gyrus granule cells in the hippocampus, and then, at an early age, moved into the cell body. Again, there were APOE allele–specific patterns to tau propagation. The team saw four major tau staining patterns, which correlated with the level of brain atrophy. Types 1 and 2, associated with least atrophy, occurred most often in the tau-only, ApoE-negative mice; type 4, associated with the greatest atrophy, occurred most often in the tau-E4 mice.
“The featured distribution of these ... patterns, which either represent different tau structures or progressively more advanced pathological tau stages, indicate ApoE affects either tau conformation or tau pathology progression,” the investigators wrote.
Greatest neurodegeneration seen with ApoE4
Tau-mediated neurodegeneration initiated levels of inflammatory response that also depended on the type of ApoE isoform. Exposure to a culture of damaged neurons and mixed glial cells caused microglia to release a flood of inflammatory cytokines that called in a host of astrocytes to kill damaged tau-E4 neurons en masse, but attacked the tau-E3, tau-E2, and tau-only strains much less. This finding indicates that “ApoE itself was directly involved in inducing neurotoxicity in tau-expressing susceptible neurons,” the team wrote.
Finally, they investigated this model of neurodegeneration in postmortem brain samples of patients with corticobasal degeneration, Pick disease, and progressive supranuclear palsy – the three most common sporadic primary tauopathies. Patients with the E4 allele showed more severe neurodegeneration and a greater interaction of tau pathology and neurodegeneration. Amyloid deposition was associated with less severe neurodegeneration.
Taken together, the findings strongly suggest that the high-risk APOE4 allele is the linchpin that links neuroinflammation to neuronal death in the setting of tau pathology, the investigators concluded.
“The presence of degenerating neurons appeared to further induce neuroinflammation, which was augmented by ApoE4 owing to its inherently higher innate immune reactivity. While activated microglia may be protective to some extent in the setting of amyloid-beta pathology, by targeting plaques and reducing dystrophic neurites, they could be deleterious in tauopathy by directly targeting injured neurons and by activating toxic astrocytes. Enhanced neuroinflammation associated with ApoE4 may further exacerbate neurodegeneration.”
The study was funded by grants awarded to multiple investigators by the National Institutes of Health, the JPB Foundation, Cure Alzheimer’s Fund, AstraZeneca, the Consortium for Frontotemporal Dementia Research, the Tau Consortium, the National Multiple Sclerosis Society, the Nancy Davis Foundation, and the Amyotrophic Lateral Sclerosis Association. Dr. Holtzman cofounded and is on the scientific advisory board of C2N Diagnostics. He consults for Genentech, AbbVie, Eli Lilly, Proclara, GlaxoSmithKline, and Denali. Dr. Holtzman’s lab is funded by institutional research grants from C2N Diagnostics, Eli Lilly, AbbVie, and Denali.
[email protected]
On Twitter @Alz_Gal
FROM NATURE
Key clinical point:
Major finding: The presence of the e4 allele was associated with brain atrophy and neurodegeneration in a transgenic mouse model, while the absence of any APOE allele was neuroprotective.
Data source: The in vivo study employed transgenic mice that expressed the human tau protein along with human ApoE variants.
Disclosures: The study was funded by grants awarded to multiple investigators by the National Institutes of Health, the JPB Foundation, Cure Alzheimer’s Fund, AstraZeneca, the Consortium for Frontotemporal Dementia Research, the Tau Consortium, the National Multiple Sclerosis Society, the Nancy Davis Foundation, and the Amyotrophic Lateral Sclerosis Association. Dr. Holtzman cofounded and is on the scientific advisory board of C2N Diagnostics. He consults for Genentech, AbbVie, Eli Lilly, Proclara, GlaxoSmithKline, and Denali. Dr. Holtzman’s lab is funded by institutional research grants from C2N Diagnostics, Eli Lilly, AbbVie, and Denali.