Faculty Bibliography

Endosomal system dysfunction within neurons is a prominent early feature of Alzheimer's disease (AD) pathology. Multiple AD risk factors are regulators of endocytosis and are known to cause hyper-activity of the early-endosome small GTPase rab5, resulting in neuronal endosomal pathway disruption and cholinergic neurodegeneration. Adaptor protein containing Pleckstrin homology domain, Phosphotyrosine binding domain, Leucine zipper motif (APPL1), an important rab5 effector protein and signaling molecule, has been shown in vitro to interface between endosomal and neuronal dysfunction through a rab5-activating interaction with the BACE1-generated C-terminal fragment of amyloid precursor protein (APP-βCTF), a pathogenic APP fragment generated within endosomal compartments. To understand the contribution of APPL1 to AD-related endosomal dysfunction in vivo, we generated a transgenic mouse model over-expressing human APPL1 within neurons (Thy1-APPL1). Strongly supporting the important endosomal regulatory roles of APPL1 and their relevance to AD etiology, Thy1-APPL1 mice (both sexes) develop enlarged neuronal early endosomes and increased synaptic endocytosis due to increased rab5 activation. We demonstrated pathophysiological consequences of APPL1 overexpression, including functional changes in hippocampal long-term potentiation (LTP) and long-term depression (LTD), degeneration of large projection cholinergic neurons of the basal forebrain, and impaired hippocampal-dependent memory. Our evidence shows that neuronal APPL1 elevation modeling its functional increase in the AD brain induces a cascade of AD-related pathological effects within neurons, including early endosome anomalies, synaptic dysfunction, and selective neurodegeneration. Our in vivo model highlights the contributions of APPL1 to the pathobiology and neuronal consequences of early endosomal pathway disruption and its potential value as a therapeutic target.Significance Statement Neuronal endosome dysfunction appears early in Alzheimer's disease (AD) and is linked to memory loss. Genes and risk factors associated with AD often increase rab5 activity, a protein that disrupts endosomal signalling when hyperactivated. APPL1, a key rab5 partner, worsens this dysfunction via its interaction with APP-βCTF, a protein fragment associated with AD. To explore APPL1's role, we created a genetically modified mouse that overexpresses APPL1 in neurons. This model provides the first in vivo evidence that APPL1 overexpression triggers key AD-like effects: rab5 hyperactivation, enlarged early endosomes, loss of cholinergic neurons, reduced synaptic plasticity in memory-related brain regions, and memory deficits. These findings highlight APPL1's role in AD pathogenesis and its potential as a therapeutic target.

Endosomal system dysfunction within neurons is a prominent early feature of Alzheimer's disease (AD) pathology. Multiple AD risk factors are regulators of endocytosis and are known to cause hyper-activity of the early-endosome small GTPase rab5, resulting in neuronal endosomal pathway disruption and cholinergic neurodegeneration. Adaptor protein containing Pleckstrin homology domain, Phosphotyrosine binding domain, Leucine zipper motif (APPL1), an important rab5 effector protein and signaling molecule, has been shown in vitro to interface between endosomal and neuronal dysfunction through a rab5-activating interaction with the BACE1-generated C-terminal fragment of amyloid precursor protein (APP-βCTF), a pathogenic APP fragment generated within endosomal compartments. To understand the contribution of APPL1 to AD-related endosomal dysfunction in vivo, we generated a transgenic mouse model over-expressing human APPL1 within neurons (Thy1-APPL1). Strongly supporting the important endosomal regulatory roles of APPL1 and their relevance to AD etiology, Thy1-APPL1 mice (both sexes) develop enlarged neuronal early endosomes and increased synaptic endocytosis due to increased rab5 activation. We demonstrated pathophysiological consequences of APPL1 overexpression, including functional changes in hippocampal long-term potentiation (LTP) and long-term depression (LTD), degeneration of large projection cholinergic neurons of the basal forebrain, and impaired hippocampal-dependent memory. Our evidence shows that neuronal APPL1 elevation modeling its functional increase in the AD brain induces a cascade of AD-related pathological effects within neurons, including early endosome anomalies, synaptic dysfunction, and selective neurodegeneration. Our in vivo model highlights the contributions of APPL1 to the pathobiology and neuronal consequences of early endosomal pathway disruption and its potential value as a therapeutic target.Significance Statement Neuronal endosome dysfunction appears early in Alzheimer's disease (AD) and is linked to memory loss. Genes and risk factors associated with AD often increase rab5 activity, a protein that disrupts endosomal signalling when hyperactivated. APPL1, a key rab5 partner, worsens this dysfunction via its interaction with APP-βCTF, a protein fragment associated with AD. To explore APPL1's role, we created a genetically modified mouse that overexpresses APPL1 in neurons. This model provides the first in vivo evidence that APPL1 overexpression triggers key AD-like effects: rab5 hyperactivation, enlarged early endosomes, loss of cholinergic neurons, reduced synaptic plasticity in memory-related brain regions, and memory deficits. These findings highlight APPL1's role in AD pathogenesis and its potential as a therapeutic target.

BACKGROUND:/calmodulin-dependent protein kinase II and selected for probe collection. RESULTS:This approach significantly reduced the amount of FFPE tissue needed for robust single population RNA-seq. We demonstrate ~20 identified L3 or L5 pyramidal neurons or one lamina-specific cortical ribbon from a single 5µm thick section is sufficient to generate robust RNA-seq reads. Bioinformatic analysis of neurons and ribbons showed notable similarities and differences reflective of the single neuron and multiple admixed cell types within the former and latter, respectively. Comparison with existing methods Protocols exist for DSP of postmortem human FFPE brain tissue. However, this new approach enables profiling small groups of ~14-21 pyramidal neurons using the GeoMx DSP platform. CONCLUSIONS:This optimized DSP assay provides high resolution RNA-seq data demonstrating utility and versatility of the GeoMx platform for individually characterized neurons and isolated cortical ribbons within postmortem FFPE human brain tissue for downstream analyses.

Huntington's disease (HD) is caused by the expansion of the polyglutamine stretch in huntingtin protein (HTT) resulting in hallmark aggresomes/inclusion bodies (IBs) composed of mutant huntingtin protein (mHTT) and its fragments. Stimulating autophagy to enhance mHTT clearance is considered a potential therapeutic strategy for HD. Our recent evaluation of the autophagic-lysosomal pathway (ALP) in human HD brain reveals upregulated lysosomal biogenesis and relatively normal autophagy flux in early Vonsattel grade brains, but impaired autolysosome clearance in late grade brains, suggesting that autophagy stimulation could have therapeutic benefits as an early clinical intervention. Here, we tested this hypothesis by crossing the Q175 HD knock-in model with our autophagy reporter mouse TRGL (Thy-1-RFP-GFP-LC3) to investigate in vivo neuronal ALP dynamics. In the Q175 and/or TRGL/Q175 mice, mHTT was detected in autophagic vacuoles and also exhibited a high level of colocalization with autophagy receptors p62/SQSTM1 and ubiquitin in the IBs. Compared to the robust lysosomal pathology in late-stage human HD striatum, ALP alterations in Q175 models are also late-onset but milder, that included a lowered phospho-p70S6K level, lysosome depletion, and autolysosome elevation including more poorly acidified autolysosomes and larger-sized lipofuscin granules, reflecting impaired autophagic flux. Administration of a mTOR inhibitor to 6-mo-old TRGL/Q175 normalized lysosome number, ameliorated aggresome pathology while reducing mHTT-, p62-, and ubiquitin-immunoreactivities, suggesting the beneficial potential of autophagy modulation at early stages of disease progression.

Epilepsy is a chronic condition characterized by unpredictable and recurrent spontaneous seizures. In a previous study, we reported that pharmacological inhibition of calpain prevented epileptogenesis in the rat pilocarpine model. In this study, we demonstrate that transgenic overexpression of calpastatin, the endogenous inhibitor of calpain, reduces calpain activation and lessens seizure burden in the mouse intrahippocampal kainate model. Blockade of calpain activation was evidenced by a reduction in the generation of spectrin breakdown products, a hallmark of calpain activation. CAST overexpression was associated with a significant reduction in seizure burden, further supporting the idea that blocking calpain overactivation prevents epilepsy. Moreover, a reduction in seizure burden was accompanied by a decrease in inflammatory markers but not cell death. Together, these observations corroborate the role of calpain overactivation in epileptogenesis and provide further support for the use of calpain inhibitors as a viable strategy to prevent epilepsy. PLAIN LANGUAGE SUMMARY: The mechanisms by which brain alterations lead to spontaneous seizures are not well understood. Acquired epilepsy often follows brain trauma. After a brain injury, the activation of the protease calpain has been associated with the development of spontaneous seizures. Our observations indicate that transgenic overexpression of calpastatin, an endogenous inhibitor of calpain, impacts epileptogenesis and reduces seizure burden. This suggests that inhibiting calpain could be a viable strategy to prevent epilepsy.

Mutations in PSEN1 were first discovered as a cause of Alzheimer's disease (AD) in 1995, yet the mechanism(s) by which the mutations cause disease still remains unknown. The generation of novel mouse models assessing the effects of different mutations could aid in this endeavor. Here we report on transgenic mouse lines made with the Δ440 PSEN1 mutation that causes AD with parkinsonism:- two expressing the un-tagged human protein and two expressing a HA-tagged version. Detailed characterization of these lines showed that Line 305 in particular, which expresses the untagged protein, develops age-dependent memory deficits and pathologic features, many of which are consistent with features found in AD. Key behavioral and physiological alterations found in the novel 305 line included an age-dependent deficit in spontaneous alternations in the Y-maze, a decrease in exploration of the center of an open field box, a decrease in the latency to fall on a rotarod, a reduction in synaptic strength and pair-pulse facilitation by electrophysiology, and profound alterations to cerebral blood flow regulation. The pathologic alterations found in the line included, significant neuronal loss in the hippocampus and cortex, astrogliosis, and changes in several proteins involved in synaptic and mitochondrial function, Ca2+ regulation, and autophagy. Taken together, these findings suggest that the transgenic lines will be useful for the investigation of AD pathogenesis.

Autophagy is a lysosome-based degradative process used to recycle obsolete cellular constituents and eliminate damaged organelles and aggregate-prone proteins. Their postmitotic nature and extremely polarized morphologies make neurons particularly vulnerable to disruptions caused by autophagy-lysosomal defects, especially as the brain ages. Consequently, mutations in genes regulating autophagy and lysosomal functions cause a wide range of neurodegenerative diseases. Here, we review the role of autophagy and lysosomes in neurodegenerative diseases such as Alzheimer disease, Parkinson disease and frontotemporal dementia. We also consider the strong impact of cellular ageing on lysosomes and autophagy as a tipping point for the late-age emergence of related neurodegenerative disorders. Many of these diseases have primary defects in autophagy, for example affecting autophagosome formation, and in lysosomal functions, especially pH regulation and calcium homeostasis. We have aimed to provide an integrative framework for understanding the central importance of autophagic-lysosomal function in neuronal health and disease.

UNLABELLED:Dysfunction of the endolysosomal system within neurons is a prominent feature of Alzheimer's disease (AD) pathology. Multiple AD-risk factors are known to cause hyper-activity of the early-endosome small GTPase rab5, resulting in neuronal endosomal pathway disruption. APPL1, an important rab5 effector protein, is an interface between endosomal and neuronal function through a rab5-activating interaction with the BACE1-generated C-terminal fragment (βCTF or C99) of the amyloid precursor protein (APP), a pathogenic APP fragment generated within endolysosomal compartments. To better understand the role of APPL1 in the AD endosomal phenotype, we generated a transgenic mouse model over-expressing human APPL1 within neurons (Thy1-APPL1 mice). Consistent with the important endosomal regulatory role of APPL1, Thy1-APPL1 mice have enlarged neuronal early endosomes and increased synaptic endocytosis due to increased rab5 activation. We additionally demonstrate pathological consequences of APPL1 overexpression, including functional changes in hippocampal long-term potentiation (LTP) and long-term depression (LTD), as well as degeneration of the large projection cholinergic neurons of the basal forebrain and impairment of hippocampal-dependent memory. Our findings show that increased neuronal APPL1 levels lead to a cascade of pathological effects within neurons, including early endosomal alterations, synaptic dysfunction, and neurodegeneration. Multiple risk factors and molecular regulators, including APPL1 activity, are known to contribute to the endosomal dysregulation seen in the early stages of AD, and these findings further highlight the shared pathobiology and consequences to a neuron of early endosomal pathway disruption. SIGNIFICANCE STATEMENT/UNASSIGNED:Dysfunction in the endolysosomal system within neurons is a key feature of Alzheimer's disease (AD). Multiple AD risk factors lead to hyperactivity of the early-endosome GTPase rab5, disrupting neuronal pathways including the cholinergic circuits involved early in memory decline. APPL1, a crucial rab5 effector, connects endosomal and neuronal functions through its interaction with a specific amyloid precursor protein (APP) fragment generated within endosomes. To understand APPL1's role, a transgenic mouse model over-expressing human APPL1 in neurons (Thy1-APPL1 mice) was developed. These mice show enlarged early endosomes and increased synaptic endocytosis due to rab5 activation, resulting in impaired hippocampal long-term potentiation and depression, the degeneration of basal forebrain cholinergic neurons, and memory deficits, highlighting a pathological cascade mediated through APPL1 at the early endosome.

Autophagy, the major lysosomal pathway for degrading damaged or obsolete constituents, protects neurons by eliminating toxic organelles and peptides, restoring nutrient and energy homeostasis, and inhibiting apoptosis. These functions are especially vital in neurons, which are postmitotic and must survive for many decades while confronting mounting challenges of cell aging. Autophagy failure, especially related to the declining lysosomal ("phagy") functions, heightens the neuron's vulnerability to genetic and environmental factors underlying Alzheimer's disease (AD) and other late-age onset neurodegenerative diseases. Components of the global autophagy-lysosomal pathway and the closely integrated endolysosomal system are increasingly implicated as primary targets of these disorders. In AD, an imbalance between heightened autophagy induction and diminished lysosomal function in highly vulnerable pyramidal neuron populations yields an intracellular lysosomal build-up of undegraded substrates, including APP-βCTF, an inhibitor of lysosomal acidification, and membrane-damaging Aβ peptide. In the most compromised of these neurons, β-amyloid accumulates intraneuronally in plaque-like aggregates that become extracellular senile plaques when these neurons die, reflecting an "inside-out" origin of amyloid plaques seen in human AD brain and in mouse models of AD pathology. In this review, the author describes the importance of lysosomal-dependent neuronal cell death in AD associated with uniquely extreme autophagy pathology (PANTHOS) which is described as triggered by lysosomal membrane permeability during the earliest "intraneuronal" stage of AD. Effectors of other cell death cascades, notably calcium-activated calpains and protein kinases, contribute to lysosomal injury that induces leakage of cathepsins and activation of additional death cascades. Subsequent events in AD, such as microglial invasion and neuroinflammation, induce further cytotoxicity. In major neurodegenerative disease models, neuronal death and ensuing neuropathologies are substantially remediable by reversing underlying primary lysosomal deficits, thus implicating lysosomal failure and autophagy dysfunction as primary triggers of lysosomal-dependent cell death and AD pathogenesis and as promising therapeutic targets.