Ceruloplasmin immunoreactivity in neurodegenerative disorders

Ceruloplasmin (CP) is a 132kd cuproprotein which, together with transferrin, provides the majority of anti-oxidant capacity in serum. Increased iron deposition and lipid peroxidation in the basal ganglia of subjects with hereditary CP deficiency suggest that CP may serve as an anti-oxidant in the brain as well. The present study compared CP immunoreactivity in brain specimens from normal controls and subjects with neurodegenerative disorders (Alzheimer's disease [AD], Parkinson's disease [PD], progressive supranuclear palsy [PSP], and Huntington's disease [HD]) (n = 5 per group). The relative intensity of neuronal CP staining and the numbers of CP-stained neurons per 25x microscope field were determined in hippocampus (CA1, subiculum, and parahippocampal gyrus), parietal cortex, frontal cortex, substantia nigra, and caudate. CP was detected in both neurons and astrocytes in all specimens, and in senile plaques and occasional neurofibrillary tangles in AD brain. Neuronal CP staining intensity tended to increase in most AD brain regions, but was statistically significant vs controls only in the CA1 region of hippocampus (p = .016). Neuronal CP staining in brain specimens from other neurodegenerative disorders showed a slight but nonsignificant increase vs controls. The numbers of CP-stained neurons per field did not differ between the various neurodegenerative disorders and controls. These results suggest that a modest increase in neuronal CP content is present in the AD brain, and lesser elevations in neuronal CP occur in the other neurodegenerative disorders in this study. Though CP functions as both an acute phase protein and an anti-oxidant in peripheral tissues, whether it does so in the brain remains to be determined.     

Immunocytochemical Detection of Anti-Hippocampal Antibodies in Alzheimer's Disease and Normal Cerebrospinal Fluid

Immunocytochemical staining was performed to investigate the presence of anti-hippocampal antibodies in cerebrospinal fluid (CSF) from patients with probable Alzheimer's disease (AD) (n = 19), aged normal controls (n = 9), and young normal controls (n = 10). Marked staining of neurons in the granule cell layer of the dentate gyrus and in pyramidal neurons in CA1-3 of the rat hippocampus was observed in 5 AD CSF samples (26%), 1 aged control sample (11%), and 1 young control sample (10%). These differences were not statistically significant. One of the immunoreactive AD CSF specimens also contained high concentrations of C5b-9, the membrane attack complex. The infrequent occurrence of anti-hippocampal antibodies in AD CSF, and the detection of similar immunoreactivity in control CSF specimens, suggest that these antibodies are unlikely to play a role in the neurodegenerative process in most individuals with AD. However, elevated C5b-9 concentration in an AD CSF specimen with marked immunoreactivity to hippocampal neurons suggests the possibility that anti-neuronal antibodies may contribute to complement activation in some AD patients.     

iTRAQ analysis of complex proteome alterations in 3xTgAD Alzheimer's mice: understanding the interface between physiology and disease

Alzheimer's disease (AD) is characterized by progressive cognitive impairment associated with accumulation of amyloid beta-peptide, synaptic degeneration and the death of neurons in the hippocampus, and temporal, parietal and frontal lobes of the cerebral cortex. Analysis of postmortem brain tissue from AD patients can provide information on molecular alterations present at the end of the disease process, but cannot discriminate between changes that are specifically involved in AD versus those that are simply a consequence of neuronal degeneration. Animal models of AD provide the opportunity to elucidate the molecular changes that occur in brain cells as the disease process is initiated and progresses. To this end, we used the 3xTgAD mouse model of AD to gain insight into the complex alterations in proteins that occur in the hippocampus and cortex in AD. The 3xTgAD mice express mutant presenilin-1, amyloid precursor protein and tau, and exhibit AD-like amyloid and tau pathology in the hippocampus and cortex, and associated cognitive impairment. Using the iTRAQ stable-isotope-based quantitative proteomic technique, we performed an in-depth proteomic analysis of hippocampal and cortical tissue from 16 month old 3xTgAD and non-transgenic control mice. We found that the most important groups of significantly altered proteins included those involved in synaptic plasticity, neurite outgrowth and microtubule dynamics. Our findings have elucidated some of the complex proteome changes that occur in a mouse model of AD, which could potentially illuminate novel therapeutic avenues for the treatment of AD and other neurodegenerative disorders.     

Neuronal localization of the TNFalpha converting enzyme (TACE) in brain tissue and its correlation to amyloid plaques

The tumor necrosis factor (TNF)-alpha converting enzyme (TACE) can cleave the cell-surface ectodomain of the amyloid-beta precursor protein (APP), thus decreasing the generation of amyloid-beta (Abeta) by cultured non-neuronal cells. While the amyloidogenic processing of APP in neurons is linked to the pathogenesis of Alzheimer's disease (AD), the expression of TACE in neurons has not yet been examined. Thus, we assessed TACE expression in a series of neuronal and non-neuronal cell types by Western blots. We found that TACE was present in neurons and was only faintly detectable in lysates of astrocytes, oligodendrocytes, and microglial cells. Immunohistochemical analysis was used to determine the cellular localization of TACE in the human brain, and its expression was detected in distinct neuronal populations, including pyramidal neurons of the cerebral cortex and granular cell layer neurons in the hippocampus. Very low levels of TACE were seen in the cerebellum, with Purkinje cells at the granular-molecular boundary staining faintly. Because TACE was localized predominantly in areas of the brain that are affected by amyloid plaques in AD, we examined its expression in a series of AD brains. We found that AD and control brains showed similar levels of TACE staining, as well as similar patterns of TACE expression. By double labeling for Abeta plaques and TACE, we found that TACE-positive neurons often colocalized with amyloid plaques in AD brains. These observations support a neuronal role for TACE and suggest a mechanism for its involvement in AD pathogenesis as an antagonist of Abeta formation.     

Transsynaptic progression of amyloid-β-induced neuronal dysfunction within the entorhinal-hippocampal network

The entorhinal cortex (EC) is one of the earliest affected, most vulnerable brain regions in Alzheimer's disease (AD), which is associated with amyloid-β (Aβ) accumulation in many brain areas. Selective overexpression of mutant amyloid precursor protein (APP) predominantly in layer II/III neurons of the EC caused cognitive and behavioral abnormalities characteristic of mouse models with widespread neuronal APP overexpression, including hyperactivity, disinhibition, and spatial learning and memory deficits. APP/Aβ overexpression in the EC elicited abnormalities in synaptic functions and activity-related molecules in the dentate gyrus and CA1 and epileptiform activity in parietal cortex. Soluble Aβ was observed in the dentate gyrus, and Aβ deposits in the hippocampus were localized to perforant pathway terminal fields. Thus, APP/Aβ expression in EC neurons causes transsynaptic deficits that could initiate the cortical-hippocampal network dysfunction in mouse models and human patients with AD.     

Cellular mechanism of U18666A-mediated apoptosis in cultured murine cortical neurons: bridging Niemann-Pick disease type C and Alzheimer's disease

Neuronal cell death can occur by means of either necrosis or apoptosis. Both necrosis and apoptosis are generally believed to be distinct mechanisms of cell death with different characteristic features distinguished on the basis of their morphological and biochemical properties. The brain is the most cholesterol-rich organ in the body but not much is known about the mechanisms that regulate cholesterol homeostasis in the brain. Recently, several clinical and biochemical studies suggest that cholesterol imbalance in the brain may be a risk factor related to the development of neurological disorders such as Niemann-Pick disease type C (NPC) and Alzheimer's disease (AD). NPC is a fatal juvenile neurodegenerative disorder characterized by premature neuronal death and somatically altered cholesterol metabolism. The main biochemical manifestation in NPC is elevated intracellular accumulation of free cholesterol caused by a genetic deficit in cholesterol trafficking. The pharmacological agent, U18666A (3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one), is a well-known class-2 amphiphile which inhibits cholesterol transport. Cells treated with this agent accumulate intracellular cholesterol to massive levels, similar to that observed in cells from NPC patients. NPC and AD have some pathological similarities which may share a common underlying cause. AD is one of the most common types of dementia affecting the elderly. However, the molecular mechanisms of neurodegeneration in NPC and AD are largely unknown. This review provides a consolidation of work done using U18666A in the past half century and focuses on the implications of our research findings on the mechanism of U18666A-mediated neuronal apoptosis in primary cortical neurons, which may provide an insight to elucidate the mechanisms of neurodegenerative diseases, particularly NPC and AD, where apoptosis might occur through a similar mechanism.     

Brain Cytochrome Oxidase in Alzheimer''s Disease

A recent demonstration of markedly reduced (-50%) activity of cytochrome oxidase (CO; complex 4), the terminal enzyme of the mitochondrial enzyme transport chain, in platelets of patients with Alzheimer's disease (AD) suggested the possibility of a systemic and etiologically fundamental CO defect in AD. To determine whether a CO deficiency occurs in AD brain, we measured the activity of CO in homogenates of autopsied brain regions of 19 patients with AD and 30 controls matched with respect to age, postmortem time, sex, and, as indices of agonal status, brain pH and lactic acid concentration. Mean CO activity in AD brain was reduced in frontal (-26%: p less than 0.01), temporal (-17%; p less than 0.05), and parietal (-16%; not significant, p = 0.055) cortices. In occipital cortex and putamen, mean CO levels were normal, whereas in hippocampus, CO activity, on average, was nonsignificantly elevated (20%). The reduction of CO activity, which is tightly coupled to neuronal metabolic activity, could be explained by hypofunction of neurons, neuronal or mitochondrial loss, or possibly by a more primary, but region-specific, defect in the enzyme itself. The absence of a CO activity reduction in all of the examined brain areas does not support the notion of a generalized brain CO abnormality. Although the functional significance of a 16-26% cerebral cortical CO deficit in human brain is not known, a deficiency of this key energy-metabolizing enzyme could reduce energy stores and thereby contribute to the brain dysfunction and neurodegenerative processes in AD.     

Modulators of cytoskeletal reorganization in CA1 hippocampal neurons show increased expression in patients at mid-stage Alzheimer's disease

During the progression of Alzheimer's disease (AD), hippocampal neurons undergo cytoskeletal reorganization, resulting in degenerative as well as regenerative changes. As neurofibrillary tangles form and dystrophic neurites appear, sprouting neuronal processes with growth cones emerge. Actin and tubulin are indispensable for normal neurite development and regenerative responses to injury and neurodegenerative stimuli. We have previously shown that actin capping protein beta2 subunit, Capzb2, binds tubulin and, in the presence of tau, affects microtubule polymerization necessary for neurite outgrowth and normal growth cone morphology. Accordingly, Capzb2 silencing in hippocampal neurons resulted in short, dystrophic neurites, seen in neurodegenerative diseases including AD. Here we demonstrate the statistically significant increase in the Capzb2 expression in the postmortem hippocampi in persons at mid-stage, Braak and Braak stage (BB) III-IV, non-familial AD in comparison to controls. The dynamics of Capzb2 expression in progressive AD stages cannot be attributed to reactive astrocytosis. Moreover, the increased expression of Capzb2 mRNA in CA1 pyramidal neurons in AD BB III-IV is accompanied by an increased mRNA expression of brain derived neurotrophic factor (BDNF) receptor tyrosine kinase B (TrkB), mediator of synaptic plasticity in hippocampal neurons. Thus, the up-regulation of Capzb2 and TrkB may reflect cytoskeletal reorganization and/or regenerative response occurring in hippocampal CA1 neurons at a specific stage of AD progression.     

Casein Kinase 2 dependent phosphorylation of eIF4B regulates BACE1 expression in Alzheimer’s disease

Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder. Increased Aβ production plays a fundamental role in the pathogenesis of the disease and BACE1, the protease that triggers the amyloidogenic processing of APP, is a key protein and a pharmacological target in AD. Changes in neuronal activity have been linked to BACE1 expression and Aβ generation, but the underlying mechanisms are still unclear. We provide clear evidence for the role of Casein Kinase 2 in the control of activity-driven BACE1 expression in cultured primary neurons, organotypic brain slices, and murine AD models. More specifically, we demonstrate that neuronal activity promotes Casein Kinase 2 dependent phosphorylation of the translation initiation factor eIF4B and this, in turn, controls BACE1 expression and APP processing. Finally, we show that eIF4B expression and phosphorylation are increased in the brain of APPPS1 and APP-KI mice, as well as in AD patients. Overall, we provide a definition of a mechanism linking brain activity with amyloid production and deposition, opening new perspectives from the therapeutic standpoint.Subject terms: Kinases, Alzheimer''s disease, Neuronal physiology, Pathogenesis     

Neuronal localization of the TNFα converting enzyme (TACE) in brain tissue and its correlation to amyloid plaques

The tumor necrosis factor (TNF)‐α converting enzyme (TACE) can cleave the cell‐surface ectodomain of the amyloid‐β precursor protein (APP), thus decreasing the generation of amyloid‐β (Aβ) by cultured non‐neuronal cells. While the amyloidogenic processing of APP in neurons is linked to the pathogenesis of Alzheimer's disease (AD), the expression of TACE in neurons has not yet been examined. Thus, we assessed TACE expression in a series of neuronal and non‐neuronal cell types by Western blots. We found that TACE was present in neurons and was only faintly detectable in lysates of astrocytes, oligodendrocytes, and microglial cells. Immunohistochemical analysis was used to determine the cellular localization of TACE in the human brain, and its expression was detected in distinct neuronal populations, including pyramidal neurons of the cerebral cortex and granular cell layer neurons in the hippocampus. Very low levels of TACE were seen in the cerebellum, with Purkinje cells at the granular‐molecular boundary staining faintly. Because TACE was localized predominantly in areas of the brain that are affected by amyloid plaques in AD, we examined its expression in a series of AD brains. We found that AD and control brains showed similar levels of TACE staining, as well as similar patterns of TACE expression. By double labeling for Aβ plaques and TACE, we found that TACE‐positive neurons often colocalized with amyloid plaques in AD brains. These observations support a neuronal role for TACE and suggest a mechanism for its involvement in AD pathogenesis as an antagonist of Aβ formation. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 40–46, 2001     

Cytoplasmic mislocalization and mitochondrial colocalization of TDP-43 are common features between normal aged and young mice

Transactive response DNA binding protein 43 (TDP-43) pathologies have been well recognized in various neurodegenerative disorders including frontotemporal lobar degeneration (FTLD), amyotrophic lateral sclerosis (ALS), and Alzheimer’s disease (AD). However, there have been limited studies on whether there are any TDP-43 alterations in normal aging. We investigated TDP-43 distribution in different brain regions in normal aged (n = 3 for 26- or 36-month-old) compared to young (n = 3 for 6- or 12-month-old) mice. In both normal aged and young mice, TDP-43 and phosphorylated TDP-43 (pTDP-43) demonstrated a unique pattern of distribution in neurons in some specific brain regions including the pontine nuclei, thalamus, CA3 region of the hippocampus, and orbital cortex. This pattern was demonstrated on higher magnification of high-resolution double fluorescence images and confocal microscopy as mislocalization of TDP-43 and pTDP-43, characterized by neuronal nuclear depletion and cytoplasmic accumulation in these brain regions, as well as colocalization between TDP-43 or pTDP-43 and mitochondria, similar to what has been described previously in neurodegenerative disorders. All these findings were identical in both normal aged and young mice. In summary, TDP-43 and pTDP-43 mislocalization from nucleus to cytoplasm and their colocalization with mitochondria in the specific brain regions are present not only in aging, but also in young healthy states. Our findings provide a new insight for the role of TDP-43 proteinopathy in health and diseases, and that aging may not be a critical factor for the development of TDP-43 proteinopathy in subpopulations of neurons.Impact statementDespite increasing evidence implicating the important role of TDP-43 in the pathogenesis of a wide range of age-related neurodegenerative diseases, there is limited study of TDP-43 proteinopathy and its association with mitochondria during normal aging. Our findings of cytoplasmic accumulation of TDP-43 that is highly colocalized with mitochondria in neurons in selective brain regions in young animals in the absence of neuronal loss provide a novel insight into the development of TDP-43 proteinopathy and its contribution to neuronal loss.     

Humanin and the Receptors for Humanin

Alzheimer’s disease (AD) is a prevalent dementia-causing neurodegenerative disease. Neuronal death is closely linked to the progression of AD-associated dementia. Accumulating evidence has established that a 24-amino-acid bioactive peptide, Humanin, protects neurons from AD-related neuronal death. A series of studies using various murine AD models including familial AD gene-expressing transgenic mice have shown that Humanin is effective against AD-related neuronal dysfunction in vivo. Most recently, it has been shown that Humanin inhibits neuronal cell death and dysfunction by binding to a novel IL-6-receptor-related receptor(s) on the cell surface involving CNTFRα, WSX-1, and gp130. These findings suggest that endogenous Humanin [or a Humanin-like substance(s)] may suppress the onset of AD-related dementia by inhibiting both AD-related neuronal cell death and dysfunction.     

Hippocampal disruptions of synaptic and astrocyte metabolism are primary events of early amyloid pathology in the 5xFAD mouse model of Alzheimer’s disease

Alzheimer’s disease (AD) is an unremitting neurodegenerative disorder characterized by cerebral amyloid-β (Aβ) accumulation and gradual decline in cognitive function. Changes in brain energy metabolism arise in the preclinical phase of AD, suggesting an important metabolic component of early AD pathology. Neurons and astrocytes function in close metabolic collaboration, which is essential for the recycling of neurotransmitters in the synapse. However, this crucial metabolic interplay during the early stages of AD development has not been sufficiently investigated. Here, we provide an integrative analysis of cellular metabolism during the early stages of Aβ accumulation in the cerebral cortex and hippocampus of the 5xFAD mouse model of AD. Our electrophysiological examination revealed an increase in spontaneous excitatory signaling in the 5xFAD hippocampus. This hyperactive neuronal phenotype coincided with decreased hippocampal tricarboxylic acid (TCA) cycle metabolism mapped by stable ¹³C isotope tracing. Particularly, reduced astrocyte TCA cycle activity and decreased glutamine synthesis led to hampered neuronal GABA synthesis in the 5xFAD hippocampus. In contrast, the cerebral cortex of 5xFAD mice displayed an elevated capacity for oxidative glucose metabolism, which may suggest a metabolic compensation in this brain region. We found limited changes when we explored the brain proteome and metabolome of the 5xFAD mice, supporting that the functional metabolic disturbances between neurons and astrocytes are early primary events in AD pathology. In addition, synaptic mitochondrial and glycolytic function was selectively impaired in the 5xFAD hippocampus, whereas non-synaptic mitochondrial function was maintained. These findings were supported by ultrastructural analyses demonstrating disruptions in mitochondrial morphology, particularly in the 5xFAD hippocampus. Collectively, our study reveals complex regional and cell-specific metabolic adaptations in the early stages of amyloid pathology, which may be fundamental for the progressing synaptic dysfunctions in AD.Subject terms: Proteomics, Alzheimer''s disease, Molecular neuroscience, Alzheimer''s disease     

Neuroendocrine-specific protein C, a marker of neuronal differentiation, is reduced in brain of patients with Down syndrome and Alzheimer's disease

Neuroendocrine-specific protein C (NSP-C) is found in neural and neuroendocrine cells and associated with the endoplasmic reticulum. Its expression was found to correlate with the degree of neuronal differentiation. As the neuropathological findings in Down syndrome (DS) includes deficits of differentiation, and we detected a downregulated sequence with 100% homology with NSP-C homolog mRNA in temporal cortex of patients with DS as well as Alzheimer's disease (AD) using differential display-polymerase chain reaction (DD-PCR), we decided to examine the protein levels of NSP-C in temporal, frontal cortex and cerebellum of DS and AD. To normalize NSP-C versus neuronal density, we also determined neuron-specific enolase (NSE) levels and calculated the ratios. NSP-C was significantly reduced in DS (temporal and frontal cortex) and AD (frontal cortex) compared to controls. The significant decrease of NSP-C in DS was even more pronounced when related to NSE levels. Impaired differentiation in DS brain may well be due to absolutely and relatively decreased NSP-C levels in temporal and frontal cortex. As NSP-C was also reduced in AD frontal cortex, NSP-C deficits in these disorders may be reflecting neurodegenerative changes rather than a primary and specific finding of DS or AD pathogenesis.     

4-hydroxynonenal and neurodegenerative diseases

The development of oxidative stress, in which production of highly reactive oxygen species (ROS) overwhelms antioxidant defenses, is a feature of many neurological diseases: ischemic, inflammatory, metabolic and degenerative. Oxidative stress is increasingly implicated in a number of neurodegenerative disorders characterized by abnormal filament accumulation or deposition of abnormal forms of specific proteins in affected neurons, like Alzheimer's disease (AD), Pick's disease, Lewy bodies related diseases, amyotrophic lateral sclerosis (ALS), and Huntington disease. Causes of neuronal death in neurodegenerative diseases are multifactorial. In some familiar cases of ALS mutation in the gene for Cu/Zn superoxide dismutase (SOD1) can be identified. In other neurodegenerative diseases ROS have some, usually not clear, role in early pathogenesis or implications on neuronal death in advanced stages of illness. The effects of oxidative stress on "post-mitotic cells", such as neurons may be cumulative, hence, it is often unclear whether oxidative damage is a cause or consequence of neurodegeneration. Peroxidation of cellular membrane lipids, or circulating lipoprotein molecules generates highly reactive aldehydes among which one of most important is 4-hydroxynonenal (HNE). The presence of HNE is increased in brain tissue and cerebrospinal fluid of AD patients, and in spinal cord of ALS patients. Immunohistochemical studies show presence of HNE in neurofibrilary tangles and in senile plaques in AD, in the cytoplasm of the residual motor neurons in sporadic ALS, in Lewy bodies in neocortical and brain stem neurons in Parkinson's disease (PD) and in diffuse Lewy bodies disease (DLBD). Thus, increased levels of HNE in neurodegenerative disorders and immunohistochemical distribution of HNE in brain tissue indicate pathophysiological role of oxidative stress in these diseases, and especially HNE in formation of abnormal filament deposites.     

A role for semaphorin 3A signaling in the degeneration of hippocampal neurons during Alzheimer's disease

Among the earliest invariant neuropathological changes in Alzheimer's disease (AD) is the degeneration of vulnerable hippocampal CA1 and subicular pyramidal neurons. Semaphorin 3A (Sema3A) is a secreted protein that functions in signaling growth cone collapse, chemorepulsion and neuronal apoptosis during early development of the central nervous system. In this report we show that accumulation of an internalized form of Sema3A is associated with degeneration of neurons in vulnerable fields of the hippocampus during AD. Accumulation of Sema3A overlaps the appearance of phosphorylated MAP1B and tau in many neurons, suggesting that Sema3A signaling at some level may be coupled to these previously identified cytoskeletal markers of neurodegeneration. Consistent with this, we isolated and partially characterized a multiprotein complex from the hippocampus of patients with AD that contains phosphorylated MAP1B, collapsin-response mediator protein 2 (CRMP-2), Plexins A1 and A2, and a processed form of Sema3A. A model is presented in which aberrant release of Sema3A from expressing neurons in the subiculum during AD results in the internalization and transport of Sema3A from this field to CA1. Within the context of the myriad of potential insults that contribute to Alzheimer's and other neurodegenerative diseases, the bioactivity of Sema3A may contribute either directly to neurodegeneration by inducing neuronal collapse, or indirectly by abrogating the recovery capabilities of adult neurons faced with these insults.     

高压氧对脑缺血再灌注海马CA_1区神经元凋亡作用的研究

目的和方法 :应用TUNEL检测技术 ,对沙土鼠前脑缺血 2 0min后再灌注 3d模型 ,用HBO治疗连续 3d。观察HBO作用下海马CA1区神经元凋亡变化 ,研讨HBO对脑缺血再灌注损伤的疗效及其机理 ,为临床应用HBO治疗疾病提供理论依据。结果 :沙土鼠脑缺血再灌注 3d后海马CA1区大量神经元凋亡 ,HBO治疗组凋亡细胞数明显减少 (P <0 .0 1) ,并以 0 .2 5MPaHBO治疗组为佳。结论 :HBO治疗对海马神经元损伤有保护作用 ,减少神经元凋亡 ,为高压氧治疗缺血性损伤的疗效机理之一     

Proteolytic cleavage of Beclin 1 exacerbates neurodegeneration

Background

Neuronal cell loss contributes to the pathology of acute and chronic neurodegenerative diseases, including Alzheimer’s disease (AD). It remains crucial to identify molecular mechanisms sensitizing neurons to various insults and cell death. To date, the multifunctional, autophagy-related protein Beclin 1 has been shown to be both necessary and sufficient for neuronal integrity in neurodegenerative models associated with protein aggregation. Interestingly, besides its role in cellular homeostasis, Beclin 1 has also been ascribed a role in apoptosis. This makes it critical to elucidate whether Beclin 1 regulates neuronal death and survival across neurodegenerative conditions independent of protein clearance. Here, we provide experimental evidence for a direct functional link between proteolytic cleavage of Beclin 1 and apoptotic neuronal cell loss in two independent models of neurodegeneration in vivo.

Methods

Proteolytic cleavage of Beclin 1 was characterized in lysates of human AD brain samples. We developed viral tools allowing for the selective neuronal expression of the various Beclin 1 forms, including Beclin 1 cleavage products as well as a cleavage-resistant form. The effect of these Beclin 1 forms on survival and integrity of neurons was examined in models of acute and chronic neurodegeneration in vitro and in vivo. Markers of neuronal integrity, neurodegeneration and inflammation were further assessed in a Kainic acid-based mouse model of acute excitotoxic neurodegeneration and in a hAPP-transgenic mouse model of AD following perturbation of Beclin 1 in the susceptible CA1 region of the hippocampus.

Results

We find a significant increase in caspase-mediated Beclin 1 cleavage fragments in brain lysates of human AD patients and mimic this phenotype in vivo using both an excitotoxic and hAPP-transgenic mouse model of neurodegeneration. Surprisingly, overexpression of the C-terminal cleavage-fragment exacerbated neurodegeneration in two distinct models of degeneration. Local inhibition of caspase activity ameliorated neurodegeneration after excitotoxic insult and prevented Beclin 1 cleavage. Furthermore, overexpression of a cleavage-resistant form of Beclin 1 in hippocampal neurons conferred neuroprotection against excitotoxic and Amyloid beta-associated insults in vivo.

Conclusions

Together, these findings indicate that the cleavage state of Beclin 1 determines its functional involvement in both neurodegeneration and neuroprotection. Hence, manipulating the cleavage state of Beclin 1 may represent a therapeutic strategy for preventing neuronal cell loss across multiple forms of neurodegeneration.

     

Decreased Brain Protein Levels of Cytochrome Oxidase Subunits in Alzheimer's Disease and in Hereditary Spinocerebella Ataxia Disorders

Abstract : Controversy exists as to the clinical importance, cause, and disease specificity of the cytochrome oxidase (CO) activity reduction observed in some patients with Alzheimer's disease (AD). Although it is assumed that the enzyme is present in normal amount in AD, no direct measurements of specific CO protein subunits have been conducted. We measured protein levels of CO subunits encoded by mitochondrial (COX I, COX II) and nuclear (COX IV, COX VIc) DNA in autopsied brain of patients with AD whom we previously reported had decreased cerebral cortical CO activity. To assess disease specificity, groups of patients with spinocerebellar ataxia type I and Friedreich's ataxia were also included. As compared with the controls, mean protein concentrations of all four CO subunits were significantly decreased (-19 to -47%) in temporal and parietal cortices in the AD group but were not significantly reduced (-12 to -17%) in occipital cortex. The magnitude of the reduction in protein levels of the CO subunits encoded by mitochondrial DNA (-42 to -47%) generally exceeded that encoded by nuclear DNA (-19 to -43%). In the spinocerebellar ataxia disorders, COX I and COX II levels were significantly decreased in cerebellar cortex (-22 to -32%) but were normal or close to normal in cerebral cortex, an area relatively unaffected by neurodegeneration. We conclude that protein levels of mitochondrial- and nuclear-encoded CO subunits are moderately reduced in degenerating but not in relatively spared brain areas in AD and that the decrease is not specific to this disorder. The simplest explanation for our findings is that CO is decreased in human brain disorders as a secondary event in brain areas having reduced neuronal activity or neuronal/synaptic elements consequent to the primary neurodegenerative process.     

Hippocampal Adaptive Response Following Extensive Neuronal Loss in an Inducible Transgenic Mouse Model

Neuronal loss is a common component of a variety of neurodegenerative disorders (including Alzheimer''s, Parkinson''s, and Huntington''s disease) and brain traumas (stroke, epilepsy, and traumatic brain injury). One brain region that commonly exhibits neuronal loss in several neurodegenerative disorders is the hippocampus, an area of the brain critical for the formation and retrieval of memories. Long-lasting and sometimes unrecoverable deficits caused by neuronal loss present a unique challenge for clinicians and for researchers who attempt to model these traumas in animals. Can these deficits be recovered, and if so, is the brain capable of regeneration following neuronal loss? To address this significant question, we utilized the innovative CaM/Tet-DT_A mouse model that selectively induces neuronal ablation. We found that we are able to inflict a consistent and significant lesion to the hippocampus, resulting in hippocampally-dependent behavioral deficits and a long-lasting upregulation in neurogenesis, suggesting that this process might be a critical part of hippocampal recovery. In addition, we provide novel evidence of angiogenic and vasculature changes following hippocampal neuronal loss in CaM/Tet-DT_A mice. We posit that angiogenesis may be an important factor that promotes neurogenic upregulation following hippocampal neuronal loss, and both factors, angiogenesis and neurogenesis, can contribute to the adaptive response of the brain for behavioral recovery.