Proteinase inhibitor alpha 1-antichymotrypsin has different expression in various forms of neuronal ceroid lipofuscinosis.

Defective proteolytic degradation is most widely maintained as the major pathogenetic factor in neuronal ceroid lipofuscinosis (NCL). The goal of the present study was to examine the expression in NCL brain tissue of one of the serine proteinase inhibitors, alpha 1-antichymotrypsin. Our study was based on previous findings of alpha 1-antichymotrypsin association with CNS amyloidoses related to amyloid beta protein deposits and our previous findings suggesting abnormal processing of amyloid beta-protein precursor (ABPP) in NCL brains. Immunocytochemical study was performed on formalin-fixed brain tissues collected from 15 NCL cases representing four different forms of the disorder and from 16 control cases comprising age-matched controls, older nondemented individuals, and Alzheimer disease cases. Our present study has shown that the expression of alpha 1-antichymotrypsin is generally higher in NCL cases than in control cases; however, it manifests in distinct variations of intensity and proportions of immunostained cells. The strongest immunoreactivity was found in the infantile form of NCL, which is characterized by a rapid clinical course and widespread tissue damage. We found no evidence of direct involvement of alpha 1-antichymotrypsin in either the ceroid lipopigment accumulation or the abnormal processing of ABPP in NCL. However, our findings may reflect the heterogeneity of the pathomechanism underlying this group of disorders and suggest that, similarly to blood circulation, alpha 1-antichymotrypsin can also represent an acute-phase protein in brain tissue.     

Age and damage induced changes in amyloid protein precursor immunohistochemistry in the rat brain

Alzheimer's disease (AD) is characterized by the extensive deposition of the 42-amino-acid β-amyloid or A4 protein in neuritic plaques and neurofibrillary tangles within the brain. This protein is liberated from the much larger amyloid protein precursor (APP). Multiple species of APP have been proposed, including several forms that contain a 56 amino acid insert sequence analogous to the Kunitz protease inhibitors. Although expression of APP mRNA is reportedly altered in AD brain and various roles for APP have been proposed, the pathogenesis of amyloid deposition and AD remains unclear. AD is also characterized by specific memory impairments associated with decreased cholinergic activity. While aging rats do not develop mature amyloid pathology, behaviorally impaired aged rats demonstrate an analogous cholinergic decline. In this study, we examined behaviorally characterized aged rats and normal young controls for changes in APP immunohistochemistry by using anti-APP antibodies, which detect N- or C-terminal regions and which distinguish APP species with or without the Kunitz protease inhibitor domain. The results show specific age- and behavior-related changes in cortical APP immunoreactivity as well as limited numbers of APP immunoreactive deposits in the aged rats. Additionally, we found that lesions of the fimbria-fornix pathway, which in part mimic the memory impairments and loss of cholinergic activity seen in AD, result in the marked accumulation of APP immunoreactive material in the region of cholinergic fiber degeneration in the hippocampus. These findings are discussed in relation to the pathogenesis of AD in humans. © 1994 Wiley-Liss, Inc.     

Follow-up study of subunit c of mitochondrial ATP synthase (SCMAS) in Batten disease and in unrelated lysosomal disorders

Immunohistochemical and biochemical studies of subunit c of mitochondrial ATP synthase (SCMAS) storage were carried out in neuronal ceroid lipofuscinosis (NCL) and in a series of unrelated inherited and acquired lysosomal disorders. In the NCL group, represented by the late infantile, early juvenile and juvenile types, SCMAS storage was generalized neurovisceral, with considerable difference in the visceral storage pattern between the types. In late infantile NCL the SCMAS storage was intensive and corresponded to the generalized, autofluorescent, uniformly curvilinear material, irrespective of the cell type affected. In both early juvenile and juvenile NCLs the SCMAS storage was strong and almost uniform in brain neurons, but did not correlate entirely with the visceral autofluorescent storage pool, being undetectable in autofluorescent storage deposits in a constant set of tissues. In the adult (Kufs) type, the brain neurons were stained with various intensity. In infantile NCL, SCMAS storage was restricted to some of the persisting neurons. In a series of inherited lysosomal enzymopathies and acquired lysosomal disorders, excessive SCMAS accumulation was found only in secondary neuronal lipopigments. It occurred as an early and more uniform phenomenon in mucopolysaccharidosis types I, II, IIIA and in polysulphatase deficiency, or as a delayed varied phenomeno in protracted variants of mucolipidosis I, Niemann-Pick types A and C, and GM2 and GM1 gangliosidoses. Neuronal ageing led to an irregular increase in immunodetectable SCMAS epitope in some neuronal lipofuscin granules. There was no evidence of significant SCMAS lysosomal accumulation in non-neural cells in the whole group, regardless of whether lipofuscin or ceroid accumulation occurred or not. The neuronal SCMAS storage is thus nosologically a common unspecific phenomenon, which is especially amplified in NCL. The specificity of the NCL storage process is shown by the fact that even lysosomes of non-neuronal cells in NCL accumulate SCMAS. Received: 31 July 1996 / Revised: 28 October 1996 / Accepted: 31 October 1996     

Bleomycin hydrolase immunoreactivity in senile plaque in the brains of patients with Alzheimer''s disease

Bleomycin hydrolase (BH), a cysteine protease belonging to the papain superfamily, is one of the candidate beta secretases. We performed immunohistochemical studies of Alzheimer's disease (AD) brains using an antibody to BH. Polyclonal antibody to BH immunostained neocortical neurons. The immunoreactivity was also found in senile plaques in AD. These results may suggest a role of BH in amyloid formation.     

Accumulation of a repulsive axonal guidance molecule RGMa in amyloid plaques: a possible hallmark of regenerative failure in Alzheimer's disease brains

J. Satoh, H. Tabunoki, T. Ishida, Y. Saito and K. Arima (2013) Neuropathology and Applied Neurobiology 39, 109–120 Accumulation of a repulsive axonal guidance molecule RGMa in amyloid plaques: a possible hallmark of regenerative failure in Alzheimer's disease brains Aims: RGMa is a repulsive guidance molecule that induces the collapse of axonal growth cones by interacting with the receptor neogenin in the central nervous system during development. It remains unknown whether RGMa plays a role in the neurodegenerative process of Alzheimer's disease (AD). We hypothesize that RGMa, if it is concentrated on amyloid plaques, might contribute to a regenerative failure of degenerating axons in AD brains. Methods: By immunohistochemistry, we studied RGMa and neogenin (NEO1) expression in the frontal cortex and the hippocampus of 6 AD and 12 control cases. The levels of RGMa expression were determined by qRT‐PCR and Western blot in cultured human astrocytes following exposure to cytokines and amyloid beta (Aβ) peptides. Results: In AD brains, an intense RGMa immunoreactivity was identified on amyloid plaques and in the glial scar. In the control brains, the glial scar and vascular foot processes of astrocytes expressed RGMa immunoreactivity, while oligodendrocytes and microglia were negative for RGMa. In AD brains, a small subset of amyloid plaques expressed a weak NEO1 immunoreactivity, while some reactive astrocytes in both AD and control brains showed an intense NEO1 immunoreactivity. In human astrocytes, transforming growth factor beta‐1 (TGFβ₁), Aβ_1–40 or Aβ_1–42 markedly elevated the levels of RGMa, and TGFβ₁ also increased its own levels. Coimmunoprecipitation analysis validated the molecular interaction between RGMa and the C‐terminal fragment β of amyloid beta precursor protein (APP). Furthermore, recombinant RGMa protein interacted with amyloid plaques in situ. Conclusions: RGMa, produced by TGFβ‐activated astrocytes and accumulated in amyloid plaques and the glial scar, could contribute to the regenerative failure of degenerating axons in AD brains.     

GM1 ganglioside regulates the proteolysis of amyloid precursor protein

Plaques containing amyloid beta-peptides (Abeta) are a major feature in Alzheimer's disease (AD), and GM1 ganglioside is an important component of cellular plasma membranes and especially enriched in lipid raft. GM1-bound Abeta (GM1/Abeta), found in brains exhibiting early pathological changes of AD including diffuse plaques, has been suggested to be involved in the initiation of amyloid fibril formation in vivo by acting as a seed. However, the role of GM1 in amyloid beta-protein precursor (APP) processing is not yet defined. In this study, we report that exogenous GM1 ganglioside promotes Abeta biogenesis and decreases sAPPalpha secretion in SH-SY5Y and COS7 cells stably transfected with human APP695 cDNA without affecting full-length APP and the sAPPbeta levels. We also observe that GM1 increases extracellular levels of Abeta in primary cultures of mixed rat cortical neurons transiently transfected with human APP695 cDNA. These findings suggest a regulatory role for GM1 in APP processing pathways.     

Beta-secretase protein and activity are increased in the neocortex in Alzheimer disease

CONTEXT: Amyloid plaques, a major pathological feature of Alzheimer disease (AD), are composed of an internal fragment of amyloid precursor protein (APP): the 4-kd amyloid-beta protein (Abeta). The metabolic processing of APP that results in Abeta formation requires 2 enzymatic cleavage events, a gamma-secretase cleavage dependent on presenilin, and a beta-secretase cleavage by the aspartyl protease beta-site APP-cleaving enzyme (BACE). OBJECTIVE: To test the hypothesis that BACE protein and activity are increased in regions of the brain that develop amyloid plaques in AD. METHODS: We developed an antibody capture system to measure BACE protein level and BACE-specific beta-secretase activity in frontal, temporal, and cerebellar brain homogenates from 61 brains with AD and 33 control brains. RESULTS: In the brains with AD, BACE activity and protein were significantly increased (P<.001). Enzymatic activity increased by 63% in the temporal neocortex (P =.007) and 13% in the frontal neocortex (P =.003) in brains with AD, but not in the cerebellar cortex. Activity in the temporal neocortex increased with the duration of AD (P =.008) but did not correlate with enzyme-linked immunosorbent assay measures of insoluble Abeta in brains with AD. Protein level was increased by 14% in the frontal cortex of brains with AD (P =.004), with a trend toward a 15% increase in BACE protein in the temporal cortex (P =.07) and no difference in the cerebellar cortex. Immunohistochemical analysis demonstrated that BACE immunoreactivity in the brain was predominantly neuronal and was found in tangle-bearing neurons in AD. CONCLUSIONS: The BACE protein and activity levels are increased in brain regions affected by amyloid deposition and remain increased despite significant neuronal and synaptic loss in AD.     

Astrocytes accumulate A beta 42 and give rise to astrocytic amyloid plaques in Alzheimer disease brains

beta-Amyloid(1-42) (A beta 42), a major component of amyloid plaques, accumulates within pyramidal neurons in the brains of individuals with Alzheimer's disease (AD) and Down syndrome. In brain areas exhibiting AD pathology, A beta 42-immunopositive material is observed in astrocytes. In the present study, single- and double-label immunohistochemistry were used to reveal the origin and fate of this material in astrocytes. Our findings suggest that astrocytes throughout the entorhinal cortex of AD patients gradually accumulate A beta 42-positive material and that the amount of this material correlates positively with the extent of local AD pathology. A beta 42-positive material within astrocytes appears to be of neuronal origin, most likely accumulated via phagocytosis of local degenerated dendrites and synapses, especially in the cortical molecular layer. The co-localization of neuron-specific proteins, alpha 7 nicotinic acetylcholine receptor and choline acetyltransferase, in A beta 42-burdened, activated astrocytes supports this possibility. Our results also suggest that some astrocytes containing A beta 42-positive deposits undergo lysis, resulting in the formation of astrocyte-derived amyloid plaques in the cortical molecular layer in brain regions showing moderate to advanced AD pathology. These astrocytic plaques can be distinguished from those arising from neuronal lysis by virtue of their smaller size, their nearly exclusive localization in the subpial portion of the molecular layer of the cerebrocortex, and by their intense glial fibrillary acidic protein immunoreactivity. Overall, A beta 42 accumulation and the selective lysis of A beta 42-burdened neurons and astrocytes appear to make a major contribution to the observed amyloid plaques in AD brains.     

Lectin histochemistry in brains with juvenile form of neuronal ceroid-lipofuscinosis (Batten disease)

Summary Defective ultilization of dolichols in the synthesis of glycoprotein leads to an accumulation of the storage, pigment ceroid lipofuscin, containing high-mannose-type glycoconjugates, in brains affected by neuronal ceroid-lipofuscinoses (NCL). We have employed lectin histochemistry to study the distribution of such compounds and the composition of other glycoconjugates in brains of patients with a juvenile form of the disease (JNCL). Concanavalin A detected the high-manose glycoconjugates in all neurons of brains with JNCL, in lipofuscin-containing neurons of aging brains and in some neurons of age-matched control brains. Three other lectins (soybean agglutinin, Peanut agglutinin and Ulex europaeus agglutinin-I) recognized sugar moities in neurons containinglipofuscin in patients only with JNCL and not in age-matched or aging brains. The results led to the conclusion, that the binding pattern of these three lectins may differentiate between storage materials of NCL and aging brains.Supported by NIH Grant No. 5P5ONS23717D. Maslinska is a visiting scientist from Medical Research Centre, Polish Academy of Science, Warsaw, Poland     

Amyloid precursor-like protein 1 accumulates in neuritic plaques in Alzheimer’s disease

The Alzheimer’s disease (AD) β-amyloid precursor protein (APP) and the amyloid precursor-like protein 1 (APLP1) and 2 (APLP2) are members of a superfamily of proteins that appear functionally related. Although APLPs are highly homologous to APP in the N- and C-terminal domains, they lack the βA4/amyloid peptide, i.e., the main constituent of neuritic plaques in AD. To assess a potential role of APLP1 in AD, we have determined its immunohistochemical distribution in human hippocampal formation, a structure which is strongly affected in AD, and compared it with APP immunoreactivity. There was a considerable overlap of APP and APLP1 regional expression patterns. Significant APLP1 immunoreactivity was observed in neuritic plaques. Large pyramidal neurons of the subiculum showed an accumulation of APLP1 protein in their dendritic compartment. Some astrocytes elicited perinuclear APLP1 staining, but this was observed in both AD and control brains. These findings raise the possibility that APLP1 may contribute to the pathogenesis of AD-associated neurodegeneration. Received: 28 July 1997 / Revised: 28 August 1997 / Accepted: 8 September 1997     

Cholesterol‐induced astrocyte activation is associated with increased amyloid precursor protein expression and processing

Cholesterol is essential for maintaining lipid raft integrity and has been regarded as a crucial regulatory factor for amyloidogenesis in Alzheimer's disease (AD). The vast majority of studies on amyloid precursor protein (APP) metabolism and amyloid β‐protein (Aβ) production have focused on neurons. The role of astrocytes remains largely unexplored, despite the presence of activated astrocytes in the brains of most patients with AD and in transgenic models of the disease. The role of cholesterol in Aβ production has been thoroughly studied in neurons and attributed to the participation of lipid rafts in APP metabolism. Thus, in this study, we analyzed the effect of cholesterol loading in astrocytes and analyzed the expression and processing of APP. We found that cholesterol exposure induced astrocyte activation, increased APP content, and enhanced the interaction of APP with BACE‐1. These effects were associated with an enrichment of ganglioside GM1‐cholesterol patches in the astrocyte membrane and with increased ROS production. GLIA 2015;63:2010–2022     

Familial occurrence of adult-type neuronal ceroid lipofuscinosis

The adult type of neuronal ceroid lipofuscinosis (NCL) occurred in a 49-year-old man and his 51-year-old sister. They showed episodic stuporous and psychotic states, mental retardation, generalized convulsions, and ichthyosis vulgaris. At autopsy the woman had excessive accumulation of lipofuscin throughout the CNS. The degree of neuronal lipopigment accumulation was very severe in the neurons of the thalamus, substantia nigra, inferior olivary nuclei, motor nuclei of the brain stem, and cerebral cortex. Mental symptoms, such as stupor, excitement, hallucinations, and delusions, were the predominant clinical manifestations and so were misdiagnosed as schizophrenia. Though the clinical diagnosis of the adult type of NCL (Kufs' disease) is difficult because of its wide variety of manifestations, symptoms such as episodic psychotic and stuporous states accompanied by convulsive disorders with mild neurologic signs may be an indication of this disease.     

A novel carboxypeptidase B that processes native beta-amyloid precursor protein is present in human hippocampus

The processing of beta-amyloid precursor protein (APP) and generation of beta-amyloid (Abeta) are associated with the pathophysiology of Alzheimer's disease (AD). As the proteases responsible for the process in the human brain have yet to be clarified, we have searched for activities capable of cleaving native brain APP in the human hippocampus. A 40-kDa protein with proteolytic activity that degrades native brain APP in vitro was purified and characterized; molecular analysis identified it as a novel protease belonging to the carboxypeptidase B (CPB) family. PC12 cells overexpressing the cDNA encoding this protease generate a major 12-kDa beta-amyloid-bearing peptide in cytosol, a peptide which has also been detected in a cell-free system using purified brain APP as substrate. Although the protease is homologous to plasma CPB synthesized in liver, it has specific domains such as C-terminal 14 amino acid residues. Western analysis, cDNA-cloning process and Northern analysis suggested a brain-specific expression of this protease. An immunohistochemical study showed that the protease is expressed in various neuronal perikarya, including those of pyramidal neurons of the hippocampus and ependymal-choroid plexus cells, and in a portion of the microglia of normal brains. In brains of patients with sporadic AD, there is decreased neuronal expression of the protease, and clusters of microglia with protease immunoreactivity associated with its extracellular deposition are detected. These findings suggest that brain CPB has a physiological function in APP processing and may have significance in AD pathophysiology.     

Amyloid beta, mitochondrial structural and functional dynamics in Alzheimer's disease

Mitochondria are the major source of energy for the normal functioning of brain cells. Increasing evidence suggests that the amyloid precursor protein (APP) and amyloid beta (Aβ) accumulate in mitochondrial membranes, cause mitochondrial structural and functional damage, and prevent neurons from functioning normally. Oligomeric Aβ is reported to induce intracellular Ca²⁺ levels and to promote the excess accumulation of intracellular Ca²⁺ into mitochondria, to induce the mitochondrial permeability transition pore to open, and to damage mitochondrial structure. Based on recent gene expression studies of APP transgenic mice and AD postmortem brains, and APP/Aβ and mitochondrial structural studies, we propose that the overexpression of APP and the increased production of Aβ may cause structural changes of mitochondria, including an increase in the production of defective mitochondria, a decrease in mitochondrial trafficking, and the alteration of mitochondrial dynamics in neurons affected by AD. This article discusses some critical issues of APP/Aβ associated with mitochondria, mitochondrial structural and functional damage, and abnormal intracellular calcium regulation in neurons from AD patients. This article also discusses the link between Aβ and impaired mitochondrial dynamics in AD.     

Apparent loss and hypertrophy of interneurons in a mouse model of neuronal ceroid lipofuscinosis: evidence for partial response to insulin-like growth factor-1 treatment.

The neuronal ceroid lipofuscinoses (NCL) are progressive neurodegenerative disorders with onset from infancy to adulthood that are manifested by blindness, seizures, and dementia. In NCL, lysosomes accumulate autofluorescent proteolipid in the brain and other tissues. The mnd/mnd mutant mouse was first characterized as exhibiting adult-onset upper and lower motor neuron degeneration, but closer examination revealed early, widespread pathology similar to that seen in NCL. We used the autofluorescent properties of accumulated storage material to map which CNS neuronal populations in the mnd/mnd mouse show NCL-like pathological changes. Pronounced, early accumulation of autofluorescent lipopigment was found in subpopulations of GABAergic neurons, including interneurons in the cortex and hippocampus. Staining for phenotypic markers normally present in these neurons revealed progressive loss of staining in the cortex and hippocampus of mnd/mnd mice, with pronounced hypertrophy of remaining detectable interneurons. In contrast, even in aged mutant mice, many hippocampal interneurons retained staining for glutamic acid decarboxylase. Treatment with insulin-like growth factor-1 partially restored interneuronal number and reduced hypertrophy in some subregions. These results provide the first evidence for the involvement of interneurons in a mouse model of NCL. Moreover, our findings suggest that at least some populations of these neurons persist in a growth factor-responsive state.     

Cathepsin D-deficient Drosophila recapitulate the key features of neuronal ceroid lipofuscinoses

Neuronal ceroid lipofuscinoses (NCLs) are a group of lysosomal storage disorders characterized pathologically by neuronal accumulation of autofluorescent storage material and neurodegeneration. An ovine NCL form is caused by a recessive point mutation in the cathepsin D gene, which encodes a lysosomal aspartyl protease. This mutation results in typical NCL pathology with neurodegeneration and characteristic neuronal storage material. We have generated a Drosophila NCL model by inactivating the conserved Drosophila cathepsin D homolog. We report here that cathepsin D mutant flies exhibit the key features of NCLs. They show progressive neuronal accumulation of autofluorescent storage inclusions, which are also positive for periodic acid Schiff and luxol fast blue stains. Ultrastructurally, the storage material is composed of membrane-bound granular electron-dense material, similar to the granular osmiophilic deposits found in the human infantile and ovine congenital NCL forms. In addition, cathepsin D mutant flies show modest age-dependent neurodegeneration. Our results suggest that the metabolic pathway leading to NCL pathology is highly conserved during evolution, and that cathepsin D mutant flies can be used to study the pathogenesis of NCLs.     

Neuroinflammation-induced acceleration of amyloid deposition in the APPV717F transgenic mouse

It has been postulated that neuroinflammation plays a critical role in the pathogenesis of Alzheimer's disease (AD). To directly test whether an inflammatory stimulus can accelerate amyloid deposition in vivo, we chronically administered the bacterial endotoxin, lipopolysaccharide (LPS), intracerebroventricularly (i.c.v.) to 2-month-old APPV717F+/+ transgenic (TG) mice, which overexpress a mutant human amyloid precursor protein (APP 717V-F) with or without apolipoprotein E (apoE) for 2 weeks. Two weeks following central LPS administration a striking global reactive astrocytosis with increased GFAP immunoreactivity was found throughout the brains of all LPS-treated wild-type and transgenic mice including the contralateral brain hemisphere. Localized microglial activation was also evident from lectin immunostaining adjacent to the cannula track of LPS-treated mice. Quantification of thioflavine-S-positive Abeta deposits revealed a marked acceleration of amyloid deposition in LPS-treated APPV717F+/+-apoE+/+ mice compared to nontreated or vehicle-treated APPV717F+/+-apoE+/+ mice (P = 0.005). By contrast, no amyloid deposits were detected by thioflavine-S staining in LPS or vehicle-treated apoE-deficient APPV717F TG mice. Our data suggest that neuroinflammation can accelerate amyloid deposition in the APPV717F+/+ mouse model of AD and that this process requires the expression of apoE.     

Dolichols in brain and urinary sediment in neuronal ceroid lipofuscinosis

Long-chain polyisoprenoid alcohols (dolichols) increase more than tenfold from age 5 to 80 years in human cerebral cortex. The dolichol content of brain from infantile, late infantile, and juvenile forms of neuronal ceroid lipofuscinosis (NCL) was significantly higher than in age-matched patients with other neurologic diseases. Significant increase of dolichols was also found in the urinary sediment in all three types of NCL patients, and this test is useful in making the diagnosis. Dolichol accumulation is the first biochemical marker of NCL and seems to parallel storage of ceroid lipofuscin.     

Distribution of Alzheimer''s disease amyloid protein precursor in normal human and rat nervous system

Alzheimer's disease and cerebral amyloid angiopathies (CAA) are clinically heterogeneous diseases, but pathogenically related by the deposition of beta A4-amyloid in the brain in the form of neuritic plaques and/or vascular infiltrates. Antibodies directed against the N-terminal region of the predicted sequence of the beta A4 amyloid protein precursor (APP) were used to investigate the cellular distribution of this protein in the brain of normal humans and rats. We found a widespread presence of APP throughout the nervous tissue, including neurons, blood vessels, meningeal membranes, choroid plexus and ependymal cells. The highest APP immunoreactivity in both species was found in neuronal cell bodies and their processes, and around blood vessels. These findings may account for the clinical, pathological and aetiological differences found among the beta A4-amyloidosis.