Pathology of Lysosomes and Lipofucsin Granules on Human Brain Cortex. A Review

Orlando Castejón, J.

Faculty of Medicine, Zulia University, Venezuela

*Correspondence to: Dr. Orlando Castejón, J., Faculty of Medicine, Zulia University, Venezuela.

Copyright © 2018 Dr. Orlando Castejón, J. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction
Primary or secondary disturbance of lysosomal function is considered one of the predominant factors in the development of brain edema in malignant neoplasia, traumatic brain injuries and ischemic nerve cell changes [1-9], experimental allergic encephalomyelitis in dogs [10]; in capillaries of traumatic injured mouse brain [11], prion encephalopathy [12], chronic hypertensive conditions [13], in several age related and nerve cell degenerative and metabolic diseases [14-35].

Breakage or increased permeability of lysosomal membrane lead to augmentation of specially neural proteases in the cytoplasm, inducing autocatalitic areas of edematous brain parenchyma and cystic formation [5], as well as diffuse degeneration of the white matter [13].

Multiple lines of evidence implicate lysosomes in a variety of pathogenic mechanisms that produce neurodegeneration [36]. Up-regulation of lysosomes has been reported in Alzheimer disease and experimental neuronal injury [24]. Alterations to the lysosomal system contribute to protein deposits associated with different types of age-related degeneration. Lysosomes are in fact highly susceptible to free radical oxidative stress in the aging brain, and in Alzheimer’s disease [29,32,37]. The expression patterns of different classes of peptidases in central nervous system tumors include the increased synthesis and secretion of lysosomal proteolitic enzymes-cathepsyns [20,38]. Glia changes, including apoptosis of astrocytes has been related with enhanced lysosomal activity [27].

The abnormal accumulation of undigested lipid and proteins within dysfunctional endosomal/lysosomal vesicle population may serve as triggers of apoptotic cell death and neurodegeneration [31]. Dysregulation of the lysosomal system is also accompanied by the accumulation of lipofuscin granules or age pigments [39]. Koike et al. (2005) [33] showed the participation of autophagy in storage of lysosomes in neurons from mouse models of neuronal ceroid-lipofuscinoses (Batten disease). Kim et al. (2006) [35] found that lysosomal proteases are involved in the generation of N-terminal huntingtin fragments.

Lysosome-associated membrane protein 1 (LAMP-1) is a glycoprotein highly expressed in lysosomal membranes. LAMP-1 expression was enhanced in neurones with granulovacuolar degeneration. LAMP-1 also occurred in microglia and multinucleated giant cells in one Alzheimer disease case in whom amyloid burden was cleared following betaA-peptide immunization, supporting the participation of lysosomes in betaA-amyloid and, probably, in hyperphosphorylated tau turnover in AD [40,41] reported increased autophagy in postmortem brains of persons with HIV-1-associated encephalitis.

Progranulin (PGRN) deficiency causes age-related neurodegenerative diseases such as frontotemporal lobar degeneration (FTLD) and neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. PGRN is involved in modulating lysosomal function [42].

According to Weiss et al. (2014) [43] autophagy is responsible for the bulk degradation of cytoplasmic contents including organelles through the lysosomal machinery. Ischemic insults increase the formation of autophagosomes and activate autophagy in the brain of neonates following hypoxia-ischemia.

It has been shown that progranulin (PGRN) deficiency causes age-related neurodegenerative diseases such as frontotemporal lobar degeneration (FTLD) and neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. Previous studies also suggested that PGRN is involved in modulating lysosomal function [42].

Lipofuscin granules, (LGs), the “age pigments”, are autofluorescent cell products from lysosomes that diverge in number and size among brain regions. Ultrastructural analysis showed that with age LGs increase in area, but not in number [44]. Brandenstein et al. (2016) [45] found lysosomal dysfunction and impaired autophagy in a novel mouse model deficient for the lysosomal membrane protein Cln7. An accumulation of autophagic and lysosomal markers in human brain tissue from patients with primary tauopathies, such as Alzheimer disease (AD), corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP), suggest a defect of the autophagosome-lysosome pathway that may contribute to the development of tau pathology [46].

Larger amount of lysosomal aggregations in schizophrenia and bipolar disorder were reported by Krause et al. (2017) [47]. Altered lysosomal localization and cytoskeleton disruption precede the neuroinflammatory pathways, axonal dystrophy and neuronal loss previously characterized in neuronal forms of Gaucher disease [48].

In the present review we analyze the abnormalities of lysosomes and the accumulation of lipofuscin pigments in the cerebral cortex of patients with congenital malformations, brain trauma, vascular anomaly, and brain tumors, using cortical biopsies processed for transmission electron microscopy.

Lysosome Abnormalities and Presence of Lipofuscin Granules in Congenital Hydrocephalus.
In congenital hydrocephalus and Arnold-Chiari malformations, edematous neurons and glial cells exhibit fragmentation of lysosomal and multivesicular body limiting membranes. Lysosomal activation in congenital hydrocephalus is evinced by the presence of areas of focal necrosis surrounding the lysosomes, and by the high electron lucent nerve cell cytosol observed in the electron micrographs [39]. (Fig. 1).

Some lysosomes show a coarse dense granulation in their matrix and associated to their outer surface, which has been interpreted as abnormal protein aggregation of released lysosomal enzymes and adsorbed cytosolic proteins. In addition, areas of focal necrosis are observed in the neighboring cytoplasmic region, due to the lysosomal activation and release of lysosomal enzymes (Fig. 2).

The coarse granulation observed in the lysosomal matrix could correspond to denatured tau, which is metabolized within lysosomes [21], ceroid lipofuscin [23], or activated micro-calpain [30].

In congenital hydrocephalus of neonate patients, presence of lipofuscin granules also are observed (Fig. 3), suggesting that lipofuscinogenesis or formation of such residual bodies is a life span process.

Lysosomal Alteration in Severe and Complicated Human Brain Trauma
In severe and complicated human brain trauma the lysosomes appear without limiting plasma membrane and exhibiting a granular dense matrix [49-51], (Fig. 4).

Lassmann et al. (1978) [52] earlier reported lysosomal enzyme activity in pleiomorphic inclusions during the late stages of myelin degradation. Griffiths (1979) [53] found acid phophatase activity, a classical marker of lysosomes, in fly retinal degenerating axons. Gatzinski et al. (1991) [54] reported a lysosomemediated degradation of axonally transported material. Liberski et al. (2005) [55] described lysosomal electron dense bodies in neuroaxonal dystrophy in prion diseases. The lysosomal damage could be in part responsible for axonal degeneration.

In edematous non-pyramidal neurons, lobulated lysosomes and lipofuscin granules are observed around the Golgi complex area (Fig. 5).

Lipofuscin granules in mature neurons of central nervous system have been reported by Cataldo et al. (1994) [56] in populations at risk to degenerate in Alzheimer’s disease. Hayase et al. (1996) [57] described lipofuscin granules in well differentiated central neurocytoma. Takanishi et al. (1997) [20] demonstrated catepsin E colocalized with cathepsin D in lipofuscin-containing lysosomes in brain stem neurons of aged rats.

Lysosomes and Lipofuscin Granules in Reactive Astrocytes in Human Brain Trauma
The cytoplasm of phagocytic astrocytes show the presence of phagolysosomes containing dark myelin debris associated to the protein and lipidic matrix. In glycogen rich-astrocytes lysosomes also displayed bizarre shapes [58] (Fig.6).

Besides, fine granular deposits stored in clear vacuolar spaces were observed in astrocytic lysosomes and lipofuscin granules [59,60] suggesting the presence of metal or metalloid compounds, such as Fe, Zn and Cu (Fig. 7).

Ferritin-like particles were found by Sbarbaty et al. (2000) [61] in cortical ischemic lesion. Iron, ferritin and hemosiderin have been found by means of analaytical electron microscopy in lysosomes and siderosomes in Alzheimer’s disease [62]. The presence of these metallic or metalloid particles accelerate further oxidative events and interfere with the proper structures and activities of the cell leading to neurodegeneration.

Some swollen astrocytes exhibit at the cell body and perivascular end-feet an accumulation of large lipofuscin granules with vacuolated lipidic components, and clusters of dense microgranules (Fig. 8).

Lipofuscin accumulation has been classically described by numerous research workers in light and electron microscopy. Goebel et al. (1995) [60] found neuronal ceroid-lipofuscinosis in progressive ataxia and blindness. Cataldo et al. (1994) [56] in degenerating neurons and extracellular space in Alzheiumer’s disease has been also reported by Ditaranto-desimone et al. (2003) [31] in pathological aging processes, and Karan et al. (2005) [64] in macular degeneration in ELOVL4 transgenic mice.

Lysosomes in Oligodendroglial Cell in Severe and Complicated Human Brain Trauma
Oligodendroglial cells associated to degenerated myelinated axons also show lysosomes with a coarse and fine dense granulations (Castejón, 2015) (Fig. 9).

Altered permeability and fragmentation of lysosomal limiting membrane could induce abnormal activity of lysosomal proteases [12,19,17,35], which suggest a potential role of these enzymes in the pathogenesis of neurodegeneration and neuronal cell death [15,18].

The fragmented limiting plasma membrane of lysosomes could be due to peroxidative stress [65-68], arachidonic acid released by membrane phospholipids under the action of Ca2+-activated phospholipase A2 [69]. Several oxygen radical species besides superoxide radicals are produced following hypoxia. Superoxide radicals have been shown to change phospholipid and protein structures. Hydroxyl radicals are the most reactive and are known to initiate lipid peroxidation and protein oxidation. Peroxidation of polyunsaturated fatty acids damages cell membranes and disrupts transmembrane ionic gradients. The products of lipid peroxidation are aldehydes, hydrocarbon gases, and other metabolites that can also cause cytotoxic and vasogenic oedema, [70-74]. Later, Yamashima et al. (2003) [30] have demonstrated calpain-mediated disruption of lysosomal membrane after ischemia.

Lipofuscin Granules in the Capillary Wall of Vascular Malformation
In a vascular anomaly, such as anomaly of anterior cerebral artery, the endothelial peripheral cytoplasm of cerebral capillaries show large lipofuscin granules, characterized by a coarse and dense granulation of protein content and large lipid droplets (Fig.10).

Swollen pericytes embedded in a thickened basement membrane also show large aggregates of lipofuscin granules [58].

According to Auer (1975, 1979) [4,5], lysosomal proteases are markedely increased after head injury followed by well known autocatalitic areas of traumatic brain edema. The early involvement of lysosomes in neurodegeneration and cell death occurs in the form of accumulation of abnormal residual bodies or to the build of modified digestion-resistant substrates within lysosomes [15]. Yamada et al. (1994) [13] suggested that chronic edema causes increased activity of lysosomal enzymes in the cerebral cortex and subcortical white matter, and that activated lysosomal enzymes take part in the developmental mechanism of cystic formation, as well as the diffuse degeneration of the white matter.

Ditaranto-Desimone et al. (2003) [31] reported that dysregulation of lysosomal system is also accompanied by the accumulation of age associated pigment, and that this lipofuscin accumulation can sensitize cells to oxidative stress and apoptotic cell death. According to these authors, ceramide accumulation correlates with the activations of caspases prior to the appearance of cell death.

The serum proteins infiltrated throughout the extracellular space in traumatic and peritumoral edema [49], could be digested by lysosomes [9], contributing to edema resolution [39]. Intraparenchymatous hemorrhages have been currently found in the traumatized brain tissue localized in the enlarged extracellular space [50]. Iron derived from hemoglobin present in the extracellular space could catalize further oxidative activity including lysosomal degradation, and promote lipid autoxidation [74].

Lysosomal Pathology in Brain Tumors
Vacuolated lysosomes and loss of limiting membrane are observed in brain tumors surrounding fragmented Golgi apparatus (Fig. 11).

Autophagy is a protein degradation system characterized by a prominent formation of double-membrane vesicles in the tumor cytoplasm. Some studies suggest that autophagy may be important in the regulation of cancer development and progression and in determining the response of tumor cells to anticancer therapy [75-79].

Concluding Remarks
In edematous non pyramidal cells, lysosomes show fragmentation of their limiting membranes and a matrix coarse dense granulation. Areas of cytoplasmic focal necrosis are observed surrounding the lysosomes suggesting the release of lysosomal enzymes. Lipofuscin granules are also observed in neonate and infant patients with congenital hydrocephalus, suggesting that lipofuscin formation is a life span process. Lysosomes coexisting with an increased amount of lipofuscin granules are observed in young and adult patients with brain trauma, and vascular anomalies. Phagocytic astrocytes and activated oligodendroglial cells show the over-all spectrum of an altered endosomal/lysomal system. Lipofuscin granules and multivesicular bodies also are distinguished in nerve cells, endothelial and pericyte cells. The role of released and activated lysosomal enzymes is discussed in relation with the cytoplasmic focal necrosis of nerve cells and the genesis of moderate and severe edema.

Acknowledgment
The present paper was carried out with the Biological Research Institute. Faculty of Medicine of Zulia University and the Castejón Foundation logistic support.

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