Anne M. Cataldo

Endocytic Pathway Abnormalities Precede Amyloid β Deposition in Sporadic Alzheimer’s Disease and Down Syndrome: Differential Effects of APOE Genotype and Presenilin Mutations

Endocytosis is critical to the function and fate of molecules important to Alzheimers disease (AD) etiology, including the beta protein precursor (betaPP), amyloid beta (Abeta) peptide, and apolipoprotein E (ApoE). Early endosomes, a major site of Abeta peptide generation, are markedly enlarged within neurons in the Alzheimer brain, suggesting altered endocytic pathway (EP) activity. Here, we show that neuronal EP activation is a specific and very early response in AD. To evaluate endocytic activation, we used markers of internalization (rab5, rabaptin 5) and recycling (rab4), and found that enlargement of rab5-positive early endosomes in the AD brain was associated with elevated levels of rab4 immunoreactive protein and translocation of rabaptin 5 to endosomes, implying that both endocytic uptake and recycling are activated. These abnormalities were evident in pyramidal neurons of the neocortex at preclinical stages of disease when Alzheimer-like neuropathology, such as Abeta deposition, was restricted to the entorhinal region. In Down syndrome, early endosomes were significantly enlarged in some pyramidal neurons as early as 28 weeks of gestation, decades before classical AD neuropathology develops. Markers of EP activity were only minimally influenced by normal aging and other neurodegenerative diseases studied. Inheritance of the epsilon4 allele of APOE, however, accentuated early endosome enlargement at preclinical stages of AD. By contrast, endosomes were normal in size at advanced stages of familial AD caused by mutations of presenilin 1 or 2, indicating that altered endocytosis is not a consequence of Abeta deposition. These results identify EP activation as the earliest known intraneuronal change to occur in sporadic AD, the most common form of AD. Given the important role of the EP in Abeta peptide generation and ApoE function, early endosomal abnormalities provide a mechanistic link between EP alterations, genetic susceptibility factors, and Abeta generation and suggest differences that may be involved in Abeta generation and beta amyloidogenesis in subtypes of AD.

Gene expression and cellular content of cathepsin D in Alzheimer's disease brain: Evidence for early up-regulation of the endosomal-lysosomal system

In Alzheimers disease brains, more than 90% of pyramidal neurons in lamina V and 70% in lamina III displayed 2- to 5-fold elevated levels of cathepsin D (Cat D) mRNA by in situ hybridization compared with neurologically normal controls. Most of these cells appeared histologically normal. The less vulnerable nonpyramidal neuron population in lamina IV had relatively normal message levels. Neuronal populations expressing more Cat D mRNA also displayed quantitatively increased Cat D immunoreactive protein. Cat D mRNA expression was only moderately increased in astrocytes. Degenerating neurons exhibited intense immunoreactivity but lowered Cat D mRNA levels. The upregulation of Cat D synthesis and accumulation of hydrolase-laden lysosomes indicate an early activation of the endosomal-lysosomal system in vulnerable neuronal populations, possibly reflecting early regenerative or repair processes. These abnormalities also represent a basis for altered regulation of amyloid precursor protein processing.

Aβ localization in abnormal endosomes: association with earliest Aβ elevations in AD and Down syndrome

Early endosomes are a major site of amyloid precursor protein (APP) processing and a convergence point for molecules of pathologic relevance to Alzheimers disease (AD). Neuronal endosome enlargement, reflecting altered endocytic function, is a disease-specific response that develops years before the earliest stage of AD and Down syndrome (DS). We examined how endocytic dysfunction is related to Abeta accumulation and distribution in early stage AD and DS. We found by ELISA and immunocytochemistry that the appearance of enlarged endosomes coincided with an initial rise in soluble Abeta40 and Abeta42 peptides, which preceded amyloid deposition. Double-immunofluorescence using numerous Abeta antibodies showed that intracellular Abeta localized principally to rab5-positive endosomes in neurons from AD brains and was prominent in enlarged endosomes. Abeta was not detectable in neurons from normal controls and was diminished after amyloid deposition in neuropathologically confirmed AD. These studies support growing evidence that endosomal pathology contributes significantly to Abeta overproduction and accumulation in sporadic AD and in AD associated with DS and may signify earlier disease-relevant disturbances of the signaling functions of endosomes.

Lysosomal abnormalities in degenerating neurons link neuronal compromise to senile plaque development in Alzheimer disease.

Antibodies to the lysosomal hydrolases, cathepsins B and D and beta-hexosaminidase A, revealed alterations of the endosomal-lysosomal system in neurons of the Alzheimer disease brain, which preceded evident degenerative changes and became marked as atrophy, neurofibrillary pathology, or chromatolysis developed. At the earliest stages of cell atrophy, hydrolase-positive lysosomes accumulated at the basal pole and then massively throughout the perikarya and proximal and proximal dendrites of affected pyramidal neurons in Alzheimer prefrontal cortex and hippocampus, far exceeding the changes of normal aging. Secondary lysosomes as well as tertiary residual bodies (lysosomes/lipofuscin) increased implying stimulated, autophagocytosis and lysosomal system activation. Less affected brain regions, such as the thalamus, displayed similar though less extensive alterations. Certain thalamic neurons exhibited a distinctive lysosome-related abnormality characterized by the presence of cell surface blebs of varying size and number filled with intense hydrolase immunoreactivity. At more advanced stages of degeneration in still intact neurons, hydrolase-positive lipofuscin, particularly in the form of abnormally large aggregates, nearly filled the cytoplasm. Similar lipofuscin aggregates were observed in abundance in the extracellular space following cell lysis and were usually associated with deposits of the beta-amyloid protein. Degenerating neurons and their processes were the major source of these aggregates within senile plaques which contained high concentrations of acid hydrolases. We have shown in previous studies that these lysosomal hydrolases in plaques are enzymatically-active. The persistence of lysosomal structures in the brain parenchyma after neurons have degenerated is a striking and potentially diagnostic feature of Alzheimer disease which has not been observed, to our knowledge, in other degenerative diseases. The lysosomal response in degenerating Alzheimer neurons represents a probable link between an early activation of the lysosomal system in at-risk, normal-appearing neurons and the end-stage contribution of lysosomes to senile plaque formation and emphasizes a slowly progressive disturbance of the lysosomal system throughout the development of Alzheimer disease.

App Gene Dosage Modulates Endosomal Abnormalities of Alzheimer's Disease in a Segmental Trisomy 16 Mouse Model of Down Syndrome

Altered neuronal endocytosis is the earliest known pathology in sporadic Alzheimers disease (AD) and Down syndrome (DS) brain and has been linked to increased Aβ production. Here, we show that a genetic model of DS (trisomy 21), the segmental trisomy 16 mouse Ts65Dn, develops enlarged neuronal early endosomes, increased immunoreactivity for markers of endosome fusion (rab5, early endosomal antigen 1, and rabaptin5), and endosome recycling (rab4) similar to those in AD and DS individuals. These abnormalities are most prominent in neurons of the basal forebrain, which later develop aging-related atrophy and degenerative changes, as in AD and DS. We also show that App, one of the triplicated genes in Ts65Dn mice and human DS, is critical to the development of these endocytic abnormalities. Selectively deleting one copy of App or a small portion of the chromosome 16 segment containing App from Ts65Dn mice eliminated the endosomal phenotype. Overexpressing App at high levels in mice did not alter early endosomes, implying that one or more additional genes on the triplicated segment of chromosome 16 are also required for the Ts65Dn endosomal phenotype. These results identify an essential role for App gene triplication in causing AD-related endosomal abnormalities and further establish the pathogenic significance of endosomal dysfunction in AD.

Lysosomal proteinase antigens are prominently localized within senile plaques of Alzheimer's disease: evidence for a neuronal origin

To investigate the role of proteolysis in amyloid formation, we studied the localization of the proteolytic enzymes, cathepsin D and cathepsin B, in the prefrontal cerebral cortex and hippocampus of human postmortem brains from patients with Alzheimers disease and from individuals free of neurological disease. In control and Alzheimer brains, cathepsin immunoreactivity within cells was localized to lysosome-related structures, which were particularly abundant in neuronal perikarya. In Alzheimer brain, cathepsin immunoreactivity was also heavily concentrated extracellularly within senile plaques. Cathepsin immunoreactivity associated with plaques was not confined to lysosomes and was distributed throughout the plaque. Isolated amyloid cores, however, were not immunostained. Cathepsin-laden perikarya of degenerating neurons were frequently seen within senile plaques and, in the more advanced stages of degeneration, cathepsin immunoreactivity was present throughout the cytoplasm. Other identified constituents of senile plaques appeared to be less significant sources of cathepsin immunoreactivity, including astrocytes, degenerating neurites, microglia and macrophages. These results demonstrate that lysosomal proteinases are major constituents of the senile plaque and that degenerating neuronal perikarya are a principal source of the cathepsin immunoreactivity. We propose that the unregulated action of extracellular cathepsins liberated from degenerating neurons may lead to abnormal processing of the amyloid precursor protein and to the formation of amyloid locally within senile plaques in Alzheimers disease.

Lysosomal system pathways: Genes to neurodegeneration in Alzheimer's disease

The identification of cathepsins in amyloid-beta plaques revealed broad dysfunction of the lysosomal system in Alzheimers disease (AD). Coinciding with the discovery that proteolysis is required to generate the Abeta-peptide, these findings heralded an era of intense investigation on proteases in neurodegeneration. This review traces lysosomal system pathology from its early characterization to its origins within two pathways leading to the lysosome, the endocytic and autophagic pathways. An understanding has grown about how these two pathways are adversely influenced by normal brain aging and by genetic and environmental risk factors for AD, resulting in increased susceptibility of neurons to injury, amyloidogenesis, and neurodegeneration.

Alzheimer’s-related endosome dysfunction in Down syndrome is Aβ-independent but requires APP and is reversed by BACE-1 inhibition

An additional copy of the β-amyloid precursor protein (APP) gene causes early-onset Alzheimer’s disease (AD) in trisomy 21 (DS). Endosome dysfunction develops very early in DS and AD and has been implicated in the mechanism of neurodegeneration. Here, we show that morphological and functional endocytic abnormalities in fibroblasts from individuals with DS are reversed by lowering the expression of APP or β-APP-cleaving enzyme 1 (BACE-1) using short hairpin RNA constructs. By contrast, endosomal pathology can be induced in normal disomic (2N) fibroblasts by overexpressing APP or the C-terminal APP fragment generated by BACE-1 (βCTF), all of which elevate the levels of βCTFs. Expression of a mutant form of APP that cannot undergo β-cleavage had no effect on endosomes. Pharmacological inhibition of APP γ-secretase, which markedly reduced Aβ production but raised βCTF levels, also induced AD-like endosome dysfunction in 2N fibroblasts and worsened this pathology in DS fibroblasts. These findings strongly implicate APP and the βCTF of APP, and exclude Aβ and the αCTF, as the cause of endocytic pathway dysfunction in DS and AD, underscoring the potential multifaceted value of BACE-1 inhibition in AD therapeutics.

Microarray analysis of hippocampal CA1 neurons implicates early endosomal dysfunction during Alzheimer’s disease progression

BACKGROUND Endocytic dysfunction and neurotrophin signaling deficits may underlie the selective vulnerability of hippocampal neurons during the progression of Alzheimers disease (AD), although there is little direct in vivo and biochemical evidence to support this hypothesis. METHODS Microarray analysis of hippocampal CA1 pyramidal neurons acquired via laser capture microdissection was performed using postmortem brain tissue. Validation was achieved using real-time quantitative polymerase chain reaction and immunoblot analysis. Mechanistic studies were performed using human fibroblasts subjected to overexpression with viral vectors or knockdown via small interference RNA. RESULTS Expression levels of genes regulating early endosomes (rab5) and late endosomes (rab7) are selectively upregulated in homogeneous populations of CA1 neurons from individuals with mild cognitive impairment and AD. The levels of these genes are selectively increased as antemortem measures of cognition decline during AD progression. Hippocampal quantitative polymerase chain reaction and immunoblot analyses confirmed increased levels of these transcripts and their respective protein products. Elevation of select rab GTPases regulating endocytosis paralleled the downregulation of genes encoding the neurotrophin receptors TrkB and TrkC. Overexpression of rab5 in cells suppressed TrkB expression, whereas knockdown of TrkB expression did not alter rab5 levels, suggesting that TrkB downregulation is a consequence of endosomal dysfunction associated with elevated rab5 levels in early AD. CONCLUSIONS These data support the hypothesis that neuronal endosomal dysfunction is associated with preclinical AD. Increased endocytic pathway activity, driven by elevated rab GTPase expression, may result in long-term deficits in hippocampal neurotrophic signaling and represent a key pathogenic mechanism underlying AD progression.

Abnormalities in Mitochondrial Structure in Cells from Patients with Bipolar Disorder

Postmortem, genetic, brain imaging, and peripheral cell studies all support decreased mitochondrial activity as a factor in the manifestation of Bipolar Disorder (BD). Because abnormal mitochondrial morphology is often linked to altered energy metabolism, we investigated whether changes in mitochondrial structure were present in brain and peripheral cells of patients with BD. Mitochondria from patients with BD exhibited size and distributional abnormalities compared with psychiatrically-healthy age-matched controls. Specifically, in brain, individual mitochondria profiles had significantly smaller areas, on average, in BD samples (P = 0.03). In peripheral cells, mitochondria in BD samples were concentrated proportionately more within the perinuclear region than in distal processes (P = 0.0008). These mitochondrial changes did not appear to be correlated with exposure to lithium. Also, these abnormalities in brain and peripheral cells were independent of substantial changes in the actin or tubulin cytoskeleton with which mitochondria interact. The observed changes in mitochondrial size and distribution may be linked to energy deficits and, therefore, may have consequences for cell plasticity, resilience, and survival in patients with BD, especially in brain, which has a high-energy requirement. The findings may have implications for diagnosis, if they are specific to BD, and for treatment, if they provide clues as to the underlying pathophysiology of BD.