Fluorescence and electron microscopic study of the tree shrew pineal organ

Summary The fine structure of the pineal gland and the pineal innervation in the tree shrew were studied by electron microscopy and glyoxylic acid-induced fluorescence microscopy respectively. The parenchymal cells consist of pinealocytes, glial cells and pigment-containing cells. The pinealocytes are characterized by the presence of granular vesicles, synaptic ribbons, electron-dense bodies and small profiles of rER with dilated cisternae. Glial cells contain light cytoplasmic bodies, lipofuscin granules, bundles of microfilaments, and elongate profiles of rER with flattened cisternae which are often stacked together with light cytoplasmic bodies; the pigment-containing cells are unique in possessing giant pigment granules in the cytoplasm. The pinealocyte/glial cell/pigment cell in tree shrew pineals may be the same cell line of parenchymal cells at different ontogenetic stages. Pigment-containing cells contain pigment granules as a prominent cytoplasmic inclusion, suggesting they are senscent in secretory function. Both pinealocytes and glial cells contain structures suggesting secretory function such as welldeveloped Golgi complex and granular vesicles. The antigonadotrophic substances may be stored in granular vesicles. The present ultrastructural study supports the conclusion that tree shrew pineal organ is an endocrine gland which is heavily innervated by adrenergic nerves and possibly by cholinergic nerves.     

Creutzfeldt-Jakob disease: A histochemical study with microdensitometric assay of some enzymes

Summary A variety of histochemical reactions were studied in a case of Creutzfeldt-Jakob disease and these were compared with normal brain. Several enzymes were assessed quantitatively using microdensitometric techniques. Neurones showed a loss of both oxidative and lysosomal enzymes. There was a raised activity in adjacent reactive glial cells. Affected cells also showed accumulation of lipofuscin pigment and loss of cytoplasmic RNA and nuclear DNA.     

Brain peptidases: Their possible neuronal and glial localization

Neuronal and glial localization of brain peptidases was investigated by means of the kainic acid (KA) lesion technique. Activities of 6 different peptidases were measured in the rat caudate-putamen (CP) and substantia nigra (SN) 2, 7 and 21 days after unilateral intra-CP injection with 2.5 micrograms of KA. As an indicator of KA lesion in CP, substance P content in both CP and SN was also determined. In addition, activities of the same peptidases in the primary and secondary glial cell cultures of fetal rats were measured and compared to those in CP homogenate. After the KA injection, prolyl endopeptidase (Pro-EP) activity was decreased in the lesioned CP and, to a lesser extent, in the ipsilateral SN. The activity of angiotensin-converting enzyme (ACE) in the lesioned CP was decreased with a complex time course, whereas a slow and progressive reduction was observed in the SN. Alanyl and leucyl aminopeptidase (Ala-AP and Leu-AP respectively) activities gave only small changes after the lesion; Ala-AP was decreased and Leu-AP was increased in the lesioned CP, while both were decreased in the SN. Dipeptidyl aminopeptidase (DAP) and arginyl endopeptidase (Arg-EP) activities were increased 5-fold in the CP 7 days after the KA injection. Their increases paralleled that of beta-glucuronidase, the lysosomal marker enzyme. Cultured glial cells contained only a trace amount of ACE activity. Ala-AP and Pro-EP activities were considerably lower in the glial culture cells than in the CP homogenate. In contrast, DAP and Arg-EP as well as lysosomal marker enzymes showed much higher activity in the former than in the latter. These results suggest that (1) Ala-AP and Pro-EP have large neuronal components, (2) ACE is preferencially localized in neurons and (3) DAP and Arg-EP are associated with glial lysosomal function. It is, therefore, concluded that at least a part of the brain peptidases are differentially localized in neurons and glia, and may be involved in specific neuronal or glial function.     

Abnormalities in spinal neurons and dorsal root ganglion cells in Tangier disease presenting with a syringomyelia-like syndrome

A woman with homozygous Tangier disease had progressive syringomyelia-like neuropathy. She died with cardiac failure at age 61. A sural nerve biopsy taken at age 60 had shown lipid storage in Schwann and interstitial cells, and a pronounced loss of unmyelinated fibers. The neurons of the L5 spinal ganglion and, to a lesser extent, all neurons of the sacral spinal cord, contained large lipid inclusions which in electron micrographs differed from those in Schwann and satellite cells. There was no storage material in glial cells. The neuronal inclusions were membrane-bound and consisted of electron-dense and electron-lucent components. There was evidence of neuronal death in the spinal ganglion, and a diameter histogram showed that small cytons had preferentially been lost. The inclusions probably were secondary lysosomes or residual bodies, and resembled giant lipofuscin granules. Nevertheless, they were uncolored and displayed weak autofluorescence as compared to the aging pigment in control ganglia. It is tentatively suggested that the syringomyelia-like neuropathy in Tangier disease represents a lysosomal storage disorder preferentially affecting small dorsal root ganglion cells.     

Ultrastructural observations on neuronal lipofuscin (age pigment) and dense bodies induced by a proteinase inhibitor,leupeptin, in rat hippocampus

The ultrastructure of lipofuscin (age pigment) and dense bodies induced by intraventricular administration of leupeptin, a cysteine proteinase inhibitor, were investigated in the neurons of rat hippocampal dentate gyrus. Four-day treatment with leupeptin (0.5 mg/day) rapidly caused a considerable accumulation of intracytoplasmic dense bodies and swelling of neuronal processes. We demonstrated, as inner structures of the pigments, that pentalaminar structure with a thickness of 12–13 nm and finely granular matrix were exactly common to the leupeptin-induced dense bodies and lipofuscin granules. Furthermore, the transitional stages from lysosomes into the dense granules were observed in the neurons of the leupeptin-treated rats. On the other hand, some morphological differences between the leupeptin-induced dense bodies and lipofuscin granules have been shown: (1) distribution in different cell types, (2) intracytoplasmic location, (3) tendencies to associate with vacuoles, and (4) electron density. The present findings suggested that the decline of the lysosomal protein degradation could play a role in lipofuscinogenesis, especially in the genesis of their electron-dense portion, but some other mechanisms might participate in the formation and accumulation of lipofuscin with aging     

Globus pallidus glial pigment and its changes with age and chronic illness in childhood

Glial lipopigment appears in the globus pallidus without accumulating in neurons (except for late adolescence) in multiple chronic childhood diseases. In this observational study, we compared the age-related development of glial pigmentation in children with the chronic illness (cystic fibrosis) and children dying acutely. A secondary goal was to search for pallidal neuronal lipopigment in childhood. We recorded pigmentation in the brains of 37 consecutive cystic fibrosis children ranging in age from 0-23 yr and in 17 controls ranging in age from 0-18 yr. We characterized the lipofuscin histochemically and used several regression models to describe the mode of deposition. We observed that in the controls, intraglial pallidal pigment accumulated in 2 forms (relatively large globules and, separately, as clusters of fine granules) at a slow rate during childhood. In cystic fibrosis, both forms of pallidal glial pigment started accumulating at a younger age and were deposited far more rapidly. There was a further increase in the rate of accumulation between 8 and 10 yr of age. We did not encounter pallidal neuronal lipofuscin at any age. These observations are consistent with 2 propositions: 1) that globus pallidus glial cells are unique in their ability to accumulate lipofuscin before it accumulates in nearby neurons; and 2) that they are particularly susceptible to some systemic effect of this chronic illness.     

Ultrastructural heterogeneity of neuronal lipofuscin in the normal human cerebral cortex

Summary The ultrastructure of the age pigment in neurons of the normal human isocortex was investigated in seven autopsies and two biopsies. In the autopsies four cortical areas of Brodmann viz. 6, 7, 11 and 17 were studied. The lipofuscin granules appear as complexes composed of lipid and pigment. These are characterized by (1) their shape, (2) frequency per neuron, (3) the relative amount of pigment and lipid in the complexes, and (4) the structure of the matrix. The neurons contain numerous varieties of lipopigment, six of which occur with great frequency and are designated as varieties 1–6. Variety 1 is found in pyramidal neurons and seems to be characteristic of long loop projectory neuronal systems. 2, is found in large non-pyramidal neurons. 3 is found in pyramidal and non-pyramidal neurons. 4, 5 and 6 are found in non-pyramidal neurons. The pigment matrix is linear in all six varieties of lipopigment. Short linear elements are present in varieties 1, 2 and 5. Long linear elements are found in varieties 3, 4, and 6. Circular whorled arrangements are found in 4. We think that these varietics of lipofuscin may signify metabolic and/or functional differences of the different types of neurons and may contribute to the biochemical classification of cortical neurons.Supported by Deutsche Forschungsgemeinschaft Schl 141/7     

Age‐related changes of cornu ammonis 1 pyramidal neurons in gerbil transient ischemia

This study reports that postischemic apoptotic cell death of the hippocampal cornu ammonis (CA) 1 neurons is delayed in aged gerbils. Age‐related changes in the process of CA1 neuronal death following transient ischemia was studied. Two groups of Mongolian gerbils were used in this study, which compared adult (4‐month‐old) and aged (24‐month‐old) animals by hematoxylin–eosin stain, in situ nick‐end labeling (TUNEL method) and electron microscopy. In the process of neuronal death, neuronal loss of the aged group was histologically less severe than that of the adult group. TUNEL‐positive cells were found on days 3–5 after ischemia in the adult group, while they were still found on day 7 in the aged group. The apoptotic process of the aged group was delayed compared to the adult group. Furthermore, lipofuscin was ultrastructurally observed inside the apoptotic body 5 days after ischemia in CA1 pyramidal neurons of the aged group. It is likely that colocalization of lysosomal enzyme cathepsin D with lipofuscin might be associated with the age‐related alteration of lysosomal system in the neurons. Altogether these data suggest that age‐related lysosomal changes might affect the apoptotic cascade process in postischernic CA1 neurons.     

Relevance of neuronal and glial NPC1 for synaptic input to cerebellar Purkinje cells

Niemann–Pick type C disease is a rare and ultimately fatal lysosomal storage disorder with variable neurologic symptoms. The disease-causing mutations concern NPC1 or NPC2, whose dysfunction entails accumulation of cholesterol in the endosomal–lysosomal system and the selective death of specific neurons, namely cerebellar Purkinje cells. Here, we investigated whether neurodegeneration is preceded by an imbalance of synaptic input to Purkinje cells and whether neuronal or glial absence of NPC1 has different impacts on synapses. To this end, we prepared primary cerebellar cultures from wildtype or NPC1-deficient mice that are glia-free and highly enriched with Purkinje cells. We report that lack of NPC1 in either neurons or glial cells did not affect the excitability of Purkinje cells, the formation of dendrites or their excitatory synaptic activity. However, simultaneous absence of NPC1 from neuronal and glial cells impaired the presynaptic input to Purkinje cells suggesting a cooperative effect of neuronal and glial NPC1 on synapses.     

The association of infantile osteopetrosis and neuronal storage disease in two brothers

Summary Neurological manifestations in infantile osteopetrosis are common and varied, and not always attributable to the skeletal pathology. An unusual association of osteopetrosis with neuronal storage of ceroid lipofuscin is reported in two infant brothers born of nonconsanguinous parents. The first child became symptomatic at age 5 days with weight loss and vomiting. He had poor head control, hypertonia, and persistent fisting, and died at age 2 months. In the second infant, the diagnosis of osteopetrosis was confirmed at age 2 days. His neurological symptoms inlcuded blindness, deafness, and recurrent seizures. The infant died at 7 months of age. In both cases, autopsy confirmed the diffuse bony sclerosis with hepatosplenomegaly and extramedullary hematopoiesis. Neuropathological examination revealed cerebral atrophy with ventricular dilation, neuronal loss, and astrogliosis. The most striking finding was widespread accumultion of neuronal ceroid lipofuscin associated with formation of axonal spheroids. The optic nerves were compressed at the optic foramina and showed loss of myelinated axons and gliosis. Rapid Golgi impregnations of neurons from the calcarine cortex in the second infant were analyzed quantitatively, showing a reduction in the total dendritic length and number of branches. The primary defect in osteopetrosis is thought to be a lysosomal dysfunction involving the monocyte cell line from which osteoclasts are derived. Thus, the association in two brothers of osteopetrosis with accumulation of neuronal ceroid lipofuscin may not be fortuitous. The neuronal storage disorder in this instance probably reflects lysosomal dysfunction.This work was presented in part at the annual meeting of the American Association of Neuropathologists, Minneapolis, Minnesota, June 1986     

Spontaneous pallido-nigral accumulation of iron pigment and spheroid-like structures in macaque monkeys

Common incidental pathologic findings in Old World monkeys are spheroid-like structures and iron pigment in the substantia nigra and globus pallidus. The occurrence of each finding correlates with the number of years monkeys have spent in captivity. The spheroids are eosinophilic and argyrophilic, but are generally PAS, iron, and luxol fast blue negative. Ultrastructurally, they consist of aggregations of dense globules and granules interspersed with membranes; normal organelles are absent. One classic spheroid with a thin myelin sheath and accumulated fibrillar material was observed. The material in spheroids is ultrastructurally distinguishable from iron pigment, which is present in glial cells, and from neuronal lipofuscin. Accumulation of spheroids and iron pigmentation may be age-related phenomena involving portions of the brain with shared anatomical and biochemical characteristics. The study of these changes may shed light on the pathogenesis of such spheroid degenerations as Hallervorden-Spatz disease.     

Neuronal endosomal/lysosomal membrane destabilization activates caspases and induces abnormal accumulation of the lipid secondary messenger ceramide

Impairment of endosomal/lysosomal functions are reported as some of the earliest changes in several age-related neurological disorders such as Alzheimer's disease. Dysregulation of the lysosomal system is also accompanied by the accumulation of age-associated pigments and several recent reports have indicated that this age-related lipofuscin accumulation can sensitize cells to oxidative stress and apoptotic cell death. In this study, we have established and evaluated an in vitro age-related pathology paradigm that models lipofuscin accumulation. Our model consists of the treatment of cultured primary mouse neurons with lysosomotropic detergents. We have observed that one of the earliest biochemical changes associated with lysosomotropic detergent-induced membrane instability is a loss of the endosomal/lysosomal proton gradient integrity, followed by an activation of sphingomyelin hydrolysis and ceramide accumulation within enlarged endosomal/lysosomal vesicles. In addition, we demonstrate that ceramide accumulation correlates with the activation of proximal procaspases-8 and -9 as well as distal caspase-3, prior to the appearance of cell death. Taken together, we propose that disturbances of the endosomal/lysosomal system, in addition to the activation of the sphingomyelinase hydrolysis cycle, play essential roles in the course of post-mitotic neuronal aging. The abnormal accumulation of undigested lipids and proteins within dysfunctional endosomal/lysosomal vesicle populations during the process of pathological aging may serve as triggers of the cell death programs that are associated with downstream neurodegeneration.     

The lipopigments in human brain tissue necroses

Summary The ceroid pigment of macrophages were subdivided into four different types according to their own color and their ability to stain with luxol fast blue. With reference to this mode a series of more than 200 brain infarcts which had happened 1 day to 56 years before death, was examined systematically. According to the results the ceroid variants do not only succeed one another chronologically but they also represent histochemically successive phases of development which correspond to the progressive auto-oxidation of unsaturated lipids. The occurrence of the different stages is chronologically defined. During this development cortical and subcortical macrophages show different speeds in the ageing process of their pigments. There is no evidence for an essential participation of proteins in the maturation of the ceroid. Not during any phase of development is the reaction pattern identical with that of neuronal and glial lipofuscin.Dedicated to Prof. Dr. Dr. Wilhelm Müller on April 17, 1980     

Neuronal autophagy in experimental Creutzfeldt-Jakob's disease

Summary We report an experimental model of Creutzfeldt-Jakob's disease (CJD) in mice leading to the formation of giant autophagic vacuoles (AV) in neurons of the cerebral cortex. These AV appear at the end of the incubation period (4–6 months post-inoculation), together with spongy changes and clinical symptoms. Autophagy, a process of intracellular digestion of cell constituents by the lysosomal compartment, is known in many cell types, where it plays a role both in the physiological turnover and in pathological processes and is involved in protein metabolism. The same also occurs in neurons. Here autophagy is known to occur in the normal state and leads to residual bodies called lipofuscin granules. An increase in lipofuscin is known to occur in human and experimental CJD. Therefore, an increase in autophagy and in AV can be expected. In our experimental model, the activation of neuronal autophagy may be related to an alteration in neuronal protein metabolism.     

Subcellular distribution of LDH isoenzymes in neuronal- and glial-enriched fractions.

The LDH isoenzymes of cell organelles (nuclei, mitochondria, microsomes, cytosol) obtained from neuronal- and glial-enriched fractions were determined by disc polyacrylamide electrophoresis and stained for enzyme activity. The quantitative estimation was made either by elution of isoenzymes or by densitometric measurements. The neuronal fraction was found to contain mainly H forms of LDH. The glial fraction also contained H forms but its M/H ratio was higher than that of the neuronal fraction. The cell organelles displayed some specificities with respect to the LDH isoenzymes distribution. The nuclei contained mainly M forms, the mitochondria H forms and the glial microsomal fraction M forms. It is suggested that the different LDH isoenzymes are related to the specific function of the different cell organelles.     

Properties of neuronal lipofuscin pigment in mice

Summary The physical and chemical properties of the lipofuscin pigment have been studied in the neurons of C57 BL/10 mice of different ages. The results indicated the presence of two types of lipofuscin with different physical and chemical properties. The properties of these two types of lipofuscin have been discussed. It appears that the two types of lipofuscin found in young and old mice differ in their distribution, stainability, solubility, enzymatic activity, and fluorescence properties. It is suggested that these represent early and late stages in lipofuscin formation of the neurons. The pigment observed in animals deprived of Vitamin E was atypical in nature and did not conform to either of the types mentioned above.Publication No. 1017 from the Basic Health Sciences Division of Emory University. Supported by Grant No. HD-04188 from the U.S. Public Health Service.     

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     

Aging in the rat medial nucleus of the trapezoid body. II. Electron microscopy

The medial nucleus of the trapezoid body (MTB), the largest cell group of the rat superior olivary complex, was studied ultrastructurally in five groups of rats ranging in age from 2 to 33 months. Relatively little aging pigment (lipofuscin) accumulates intraneuronally, even in rats aged 33 months. The heaviest accumulation of age pigment is in glial and perivascular cells of the MTB. In rats aged 24-33 months, there is evidence of axonal and dendritic degeneration in the MTB neuropil. Aged rats also show evidence of degeneration of nerve endings. This includes the loss of calycine axosomatic terminals synapsing with MTB principal cells. All of the ultrastructural changes observed in the MTB gradually increase in frequency with advancing age.     

Effects of Acute and Chronic Phenytoin on the Electrolyte Content and the Activities of Na+, K+-, Ca2+, Mg2+ -, and HCO3--ATPases and Carbonic Anhydrase of Neonatal and Adult Rat Cerebral Cortex

Various parameters of anion and cation transport were measured in the cerebral cortex of neonatal (3-day-old) and adult rats following acute and chronic treatment with phenytoin (PHT). Acutely, PHT significantly inhibited the enzyme Na+, K+-ATPase in both neonatal and adult rats. This effect was accompanied by a significant increase in cerebral cortical Na+ content and a decrease in K+ content only in neonatal animals. Chronic treatment (two and four times a day for 7 days) of adult rats with PHT significantly reduced Na+ content without affecting whole homogenate Na+, K+-ATPase activity. The activity of this enzyme was markedly increased in the myelin- (glial product) and slightly decreased in the synaptosomal- (neuronal) fractions following chronic (four times a day for 7 days) PHT treatment. These results suggest that PHT differentially affects the two forms (neuronal and glial) of the enzyme Na+, K+-ATPase. The possible relevance of this hypothesis in relationship to the anticonvulsant and excitatory properties of PHT is discussed. Chronic (two and four times a day for 7 days) PHT treatment increased both DNA content and activity of the glial marker enzyme carbonic anhydrase. Activity of the mitochondrial enzyme HCO3- -ATPase was also increased following chronic PHT treatment. These two enzymes are intimately involved in the regulation of HCO3- -Cl- transport across glial cell and mitochondrial membranes, and these results suggest that PHT is able to affect beneficially glial regulatory processes. The ability to enhance glial regulation of anions and cations in extracellular fluid provides new and important insights into the mechanism of the anticonvulsant action of PHT.