Distribution of tripeptidyl peptidase I in human tissues under normal and pathological conditions.

Tripeptidyl peptidase I (TPP I) is a lysosomal exopeptidase that cleaves tripeptides from the free N-termini of oligopeptides. Mutations in this enzyme are associated with the classic late-infantile form of neuronal ceroid lipofuscinosis (CLN2), an autosomal recessive disorder leading to severe brain damage. To gain more insight into CLN2 pathogenesis and the role of TPP I in human tissues in general, we analyzed the temporal and spatial distribution of TPP I in the brain and its localization in internal organs under normal and pathological conditions. We report that TPP I immunoreactivity appears in neurons late in gestation and increases gradually in the postnatal period, matching significantly the final differentiation and maturation of neural tissue. Endothelial cells, choroid plexus, microglial cells, and ependyma showed TPP I immunostaining distinctly earlier than neurons. Acquisition of the adult pattern of TPP I distribution in the brain at around the age of 2 years correlates with the onset of clinical signs in CLN2 subjects. In adults, TPP I was found in all types of cells in the brain and internal organs we studied, although the intensity of TPP I labeling varied among several types of cells and showed a noticeable predilection for cells and/or organs associated with peptide hormone and neuropeptide production. In addition, TPP I immunoreactivity was increased in aging brain, neurodegenerative and lysosomal storage disorders, and some differentiated neoplasms and was reduced in ischemic/anoxic areas and undifferentiated tumors. These findings suggest that TPP I is involved in general protein turnover and that its expression may be controlled by various regulatory mechanisms, which highlights the importance of this enzyme for normal function of cells and organs in humans.

Prevention of quinolinic acid neurotoxicity in rat hippocampus in vitro by zinc. Ultrastructural observations.

The effect of zinc on the development and evolution of quinolinic acid-induced alterations in the rat hippocampus in culture was studied ultrastructurally. Zinc, although it possesses intrinsic cytotoxic properties, after application in concentrations comparable with those encountered in vivo, was able to prevent typically observed responses after quinolinic acid exposure, either early or late damage to hippocampal neurons. The results further support the concept of a potential protective effect of zinc against the neurotoxicity of particular excitotoxins.

Topographic variabilities of immunoreactivity to subunit c of mitochondrial ATP synthase and lectin binding in late infantile neuronal ceroid-lipofuscinosis.

A subset of lipophilic neurons in the brain tissue of late infantile neuronal ceroid lipofuscinosis (LINCL) cases shows in addition to finely granular storage lipopigment, larger spheroidal lysosomal inclusions, so called protein-type myoclonus bodies. Their incidence, significance, and biochemical composition have not been determined. To further characterize this type of lysosomal storage material, immunocytochemistry to subunit c of mitochondrial ATP synthase at the light and electron microscopy level, electron microscopy, and lectin histochemistry were applied. The majority of spheroidal inclusions were nonreactive to subunit c, the main protein component of the storage material in LINCL. These inclusions also showed no binding sites for the eight lectins examined, although six of the lectins used labeled finely granular storage material. According to electron and immunoelectron microscopy, spheroidal inclusions were composed of more homogeneous and more densely arranged material than typical curvilinear profiles, with shorter membranous profiles and sometime filamentous structures. The dissimilarities disclosed between finely granular lipopigment with curvilinear profiles and spheroidal inclusions in LINCL brain tissue suggest that either protein(s) other than subunit c are present in spheroidal inclusions, or subunit c in these sites undergoes conformational or proteolytic changes. These changes require further biochemical evaluations.

Rapid detection of subunit c of mitochondrial ATP synthase in urine as a diagnostic screening method for neuronal ceroid-lipofuscinoses.

Using Western blot analysis and the ELISA technique, we showed previously significantly higher levels of subunit c in the urine of individuals with late-infantile neuronal ceroid lipofuscinosis (LINCL) and some patients with juvenile NCL (JNCL) [Wisniewski et al., J. Inherited Metab Dis 17: 205-210, 1994]. In an attempt to develop a diagnostic screening test for NCL based on detection of this biochemical marker in urine, we analyzed, using the blotting technique, urine from 7 infantile NCL (INCL), 17 LINCL, and 19 JNCL cases, 30 obligate heterozygotes, and 60 control cases. This analysis confirmed our former data showing significantly higher levels of subunit c in the urine from all LINCL and some JNCL cases. No false positive results were found. This simple analytical method may serve as a fast, non-invasive screening test for NCL.

Variability in the clinical and pathological findings in the neuronal ceroid lipofuscinoses: review of data and observations.

We reviewed the clinical and pathological data on 319 neuronal ceroid lipofuscinosis (NCL) cases to determine the degree of variability within the different forms and among and within families. Thirty-six cases (11.3%) were the infantile form; 116 cases (36.3%), late infantile; 163 cases (51.1%), juvenile; and four cases (1.3%), the adult form (Kufs disease). Clinical variability was found in all forms studied, but was most striking in the juvenile and late infantile forms of NCL. The expected initial findings of seizures, dementia, blindness, or motor impairment were evident in 255 cases (80%), and rarer, less typical initial neurological symptoms were seen mainly in the 64 cases (20%) of the juvenile form: behavior abnormalities (18/64), psychoses (12/64), neuropathy (2/64), involuntary movements (15/64), ataxia (9/64). Six juvenile and two adult cases had no detectable impairment of vision. All 319 NCL cases had skin or conjunctive biopsies or buffy coats that showed the characteristic ultrastructural abnormalities of NCL. Variability was evident in 16.7% in that a combination of fingerprint, curvilinear, and membranous profile inclusion bodies was observed in storage lysosomes, although one type of inclusion was distinctly predominant for each form. Postmortem examination of brains of 19 NCL cases (three with the infantile form, six with the late infantile form, nine with the juvenile form, and one with the adult form) revealed characteristic changes. Sixteen of the 19 NCL brains (84%) showed pathological variability in that they contained more than one kind of characteristic inclusion body in the neuronal lysosomal storage compartment. In all 19 NCL brains, small amounts of aging lipofuscin were also found.(ABSTRACT TRUNCATED AT 250 WORDS)

Deposition of apolipoproteins E and J in senile plaques is topographically determined in both Alzheimer's disease and Down's syndrome brain.

The link between the immunolocalization of apolipoproteins E (apo E) and J (apo J) and the different severity of beta-amyloid deposition in various areas of Alzheimer's disease (AD) and Down's syndrome (DS) brain was analyzed. Both apolipoproteins were found in all types of senile plaques (SPs) in the cerebral cortex, which is early and severely involved in beta-amyloidosis, but apo E was seen more often than apo J in diffuse A beta deposits, especially in young DS cases and nondemented elderly persons. In the striatum and cerebellum, which show predominance of diffuse A beta deposits throughout the lifespan, apo J was absent, except for few compact deposits, whereas apo E was more widely distributed, apart from diffuse plaques in the striatum. By immunoelectron microscopy, A beta fibrils were disclosed in diffuse plaques in all brain regions studied, but not all of these early fibrillar deposits, even in the neocortex of young DS cases, showed apo E and apo J labeling. Thus, our data indicate that the immunoreactivity to apo E and J within A beta deposits is topographically determined in both AD and DS brain. Moreover, although it appears that neither of apolipoproteins studied is necessary to initiate A beta fibrillogenesis, disclosed topographic dissimilarities of their distribution within parenchymal A beta deposits suggest that they may be involved in different ways in the pathogenesis of beta-amyloidosis.

Complete cerebral ischemia with short-term survival in rats induced by cardiac arrest. I. Extracellular accumulation of Alzheimer's beta-amyloid protein precursor in the brain.

The distribution of beta-amyloid protein precursor (APP) was investigated immunocytochemically in rats subjected to global cerebral ischemia (GCI) induced by cardiac arrest. Rats underwent 10 min of GCI with 3, 6, and 12 h and 2 and 7 days of survival. APP immunostaining was found extracellular and intracellularly. Multiple extracellular APP immunoreactive deposits around and close to the vessels appeared as soon as 3 h after GCI. Extracellular accumulation of APP occurred frequently in the hippocampus, cerebral and cerebellar cortex, basal ganglia and thalamus and rarely in the brain stem. These deposits were labelled with antibodies against the N-terminal, beta-amyloid peptide, and C-terminal domains of APP. Our data suggests that either proteolytically cleaved fragments of the full-length APP or the entire APP molecule accumulates extracellularly after GCI. This findings may not only implicate the participation of APP in postischemic tissue damage but also suggest the involvement of pathomechanisms operating in ischemia in Alzheimer's disease pathology.

Complete cerebral ischemia with short-term survival in rat induced by cardiac arrest. II. Extracellular and intracellular accumulation of apolipoproteins E and J in the brain.

The distribution of apolipoprotein E (apo E) and apolipoprotein J (apo J) was investigated immunocytochemically in rats at various time intervals after 10 min global cerebral ischemia (GCI) induced by cardiac arrest. Strong apo E and weaker apo J immunoreactivity was found extracellularly in multiple deposits located close to the microvessels. These deposits appeared 3 h after GCI and were present, but not in all the animals, at all time intervals studied post-GCL. In some rats, apo E immunoreactivity was also found in small necrotic foci. Widespread, neuronal apo E immunostaining appeared 6 h post-GCI. However, the strongest neuronal apo E immunoreactivity was found 7 days post-GCI in those neurons, most often observed in the CA1 hippocampal region, exhibiting signs of ischemic cell damage. These ischemically damaged neurons displayed weaker immunoreactivity to apo J, despite its increase in the response to GCI in the various brain regions examined. Our data show that mechanisms operating in ischemia are able to supply large amounts of apo E and apo J to the brain tissue and suggest involvement of both apo E and apo J in a complex series of events occurring in the ischemic brain. Perivascular deposits of apo E/apo J colocalized with amyloid beta protein precursor epitopes that have been disclosed by us previously in this model. Whether this phenomenon is limited to postischemic brain tissue, or can be encountered also in other pathological conditions will require further elaboration.

Ubiquitin expression in globose tangles in the locus coeruleus in brains of patients with Alzheimer's and Parkinson's diseases.

The locus coeruleus belongs to brain areas exhibiting remarkable neuronal loss already during physiological aging and very early neurofibrillary tangles. We performed a semiquantitative, immunocytochemical study focused on the expression of ubiquitin in neurofibrillary tangles in the locus coeruleus as compared with the central superior nucleus. The locus coeruleus exhibited later ubiquitin expression in the neurofibrillary tangles, a lower percentage of ubiquitinated tangles and more frequently granular cytoplasmic staining with Tau-1 than the central superior nucleus. These results suggesting different, probably delayed, processing of Tau protein in the locus coeruleus might contribute additionally to cell injury in this area.

Early amyloid-beta deposits show different immunoreactivity to the amino- and carboxy-terminal regions of beta-peptide in Alzheimer's disease and Down's syndrome brain.

That the topography, severity, and progression of beta-amyloid deposition in the brain of Alzheimer's disease (AD) and Down's syndrome (DS) cases is not uniform is well documented. We have addressed at present, the issue of whether the structural composition of beta-peptide (A beta) within the early amyloid deposits might contribute to this phenomenon. The cerebral cortex, the caudate/putamen, and the cerebellum from 10 AD and 8 DS cases were immunostained with antibodies that recognize the 1-17; 17-24 amino acid residues of A beta, and the COOH-terminus of A beta 42 variant, thus to the epitopes of A beta located amino- and carboxy-terminally to the site of the putative alpha-secretase cleavage. We demonstrate that numerous diffuse, early plaques in AD and especially in DS cases show predominance of the carboxy-terminally located epitopes of A beta; the most prominent in the cerebellum, less pronounced in the cerebral cortex, and only marginal, or absent in the striatum, except for some DS cases. These data suggest that the deposition of the carboxy-terminal fragment of A beta truncated at the position of alpha-secretase cleavage or close to it in diffuse plaques may be brain-region-specific, reflecting either dissimilar processing of amyloid precursor protein or the resolution of early A beta deposits, and may substantially contribute to different progression of beta-amyloidosis in various brain regions.

Increased expression of subunit c of mitochondrial ATP synthase in brain tissue from neuronal ceroid lipofuscinoses and mucopolysaccharidosis cases but not in long-term fibroblast cultures.

Recent data showed storage of subunit c of mitochondrial ATP synthase in late infantile, juvenile, and adult forms of neuronal ceroid lipofuscinosis (NCL). The present study demonstrates that the expression of subunit c in NCL fibroblasts in long-term cultures, both grown in standard conditions and after leupeptin and ammonium chloride treatment, is not greater than in controls. It indicates that as a result of yet undefined factors, NCL fibroblasts in long-term cultures, lose their ability to accumulate subunit c. Moreover, both Western blot analysis of brain tissue homogenates and immunohistochemistry showed increased immunoreactivity to subunit c in mucopolysaccharidosis type I and III. This increased subunit c expression in a disorder with impaired lysosomal function other than the NCL supports the hypothesis that accumulation of this proteolipid might be related to its defective degradation.

Altered amyloid beta-protein precursor processing in brains of patients with neuronal ceroid lipofuscinosis.

Our previous study disclosed strong immunostaining of brain tissue in neuronal ceroid lipofuscinosis (NCL) with antibodies against amyloid beta-protein and the presence of 31-kDa polypeptide in the storage material. In the present study, we investigated the immunoreactivity of the NCL brain tissue with anti-serum (anti-GID) raised against a synthetic peptide, based on the amyloid beta-protein precursor, with the 175-186 amino acid sequence. Immunocytochemistry was performed on autopsy brain material collected from 15 NCL cases, and from 8 age-matched normal controls. The results showed strong immunoreactivity of nerve cells in the NCL cases, which according to densitometry was 5 times more intense than in the control group (P less than 0.0001 by Student's t-test). Western blot analysis revealed that in protein fractions of NCL brain homogenates anti-GID recognized the protein band of 35 kDa. Thus our present and previously performed studies supplied for the first time data pointing to abnormal processing of amyloid beta-protein precursor in NCL. Moreover, the accumulation of both 31- and 35-kDa polypeptides that was demonstrated provides further support for postulated defective protein metabolism in this disorder.

Regional differences in apolipoprotein E immunoreactivity in diffuse plaques in Alzheimer's disease brain.

Apolipoprotein E (Apo E) has been shown to be closely associated with beta amyloid in Alzheimer's disease (AD) brain. In the present study, we have found strong Apo E immunoreactivity in the amyloid cores of senile plaques (SP) in the various brain regions examined. However, Apo E immunoreactivity in diffuse plaques varied distinctly and was strong within numerous cerebellar and cortical diffuse plaques, and absent or very weak within diffuse plaques in the striatum/thalamus. This distribution of Apo E immunoreactivity in SP correlates with the occurrence of small amounts of fibrillar amyloid in diffuse plaques that has been described in the cerebral and cerebellar cortex, but not in the basal ganglia. These results show that Apo E may be associated with sites of beta amyloid fibril formation in diffuse plaques in AD brain, but they also suggest that factors other than Apo E, probably local, may influence fibrillogenesis.

Tripeptidyl-peptidase I in neuronal ceroid lipofuscinoses and other lysosomal storage disorders.

The classic late infantile form of neuronal ceroid lipofuscinosis (CLN2, cLINCL) is associated with mutations in the gene encoding tripeptidyl-peptidase I (TPP-I), a lysosomal aminopeptidase that cleaves off tripeptides from the free N-termini of oligopeptides. To date over 30 different mutations and 14 polymorphisms associated with CLN2 disease process have been identified. In the present study, we analysed the molecular basis of 15 different mutations of TPP-I by using immunocytochemistry, immunofluorescence, Western blotting, enzymatic assay and subcellular fractionation. In addition, we studied the expression of TPP-I in other lysosomal storage disorders such as CLN1, CLN3, muccopolysaccharidoses and GM1 and GM2 gangliosidoses. Our study shows that TPP-I is absent or appears in very small amounts not only in cLINCL subjects with mutations producing severely truncated protein, but also in individuals with missense point mutations, which correlates with loss of TPP-I activity. Of interest, small amounts of TPP-I were detected in lysosomal fraction from fibroblasts from cLINCL subject with protracted form. This observation suggests that the presence of small amounts of TPP-I in lysosomes is able to delay significantly CLN2 disease process. We also show that TPP-I immunoreactivity is increased in the brain tissue of CLN1 and CLN3 subjects, stronger in glial cells and macrophages than neurons. Less prominent increase of TPP-I staining was found in muccopolysaccharidoses and GM1 and GM2 gangliosidoses. These data suggest that TPP-I participates in lysosomal turnover of proteins in pathological conditions associated with cell/tissue injury.

CLN3 disease process: missense point mutations and protein depletion in vitro.

Although the CLN3 gene associated with the disease process in subjects with the juvenile form of neuronal ceroid lipofuscinosis was discovered in 1995, our knowledge of the physiological function of its gene product, CLN3 protein, is still incomplete. To gain more insight into the structural properties and function of CLN3 protein we studied at present: i) how the naturally occurring point mutations Arg334Cys and Leu101Pro affect the biological properties of CLN3 protein, and ii) whether depletion of CLN3 protein synthesis by using an antisense approach induces a distinct phenotype in cells of neuronal origin in vitro. Here we report that although both CLN3 mutant proteins are targeted to lysosomes, thus similar to wild-type CLN3 protein, they are devoid of the biological activity of wild-type CLN3 protein such as its effect on lysosomal pH or intracellular processing of amyloid-beta protein precursor and cathepsin D in vitro. The Leu101Pro mutation affected significantly the maturation and stability of CLN3 protein. The Arg334Cys mutation influenced mildly the maturation and turnover of CLN3 protein, but at the same time abolished the function of CLN3 protein in vitro, which suggests that the Arg334 may constitute a part of the active site of CLN3 protein. In addition, we show that depletion of CLN3 protein synthesis in human neuroblastoma cells in vitro induces outgrowth of long cellular processes and formation of cellular aggregates and affects the viability of these cells. This finding suggests that CLN3 protein is implicated in biological processes associated with the differentiation of cells of neuronal origin.

Atypical late infantile and juvenile forms of neuronal ceroid lipofuscinosis and their diagnostic difficulties.

We have collected 122 late-infantile neuronal ceroid lipofuscinosis (LINCL, CLN2) and 191 juvenile NCL (JNCL, CLN3) cases, diagnosed on the basis of age-at-onset, clinical symptomatology, and pathological findings and representing the most common forms of NCL in the United States, and Europe. However, careful analysis of available data revealed that about 80% of cases show typical and 20% show atypical clinical course and/or pathological findings and thus, may represent variants of LINCL and JNCL, respectively. Recent progress in the biochemistry and molecular genetics of NCL inclined us to reevaluate these atypical NCL cases. The gene responsible for LINCL has not yet been identified, except for the Finnish variant. Accumulation of subunit c of mitochondrial ATP synthase, to curvilinear profiles, is found in LINCL cases. A novel variant of LINCL, with predominantly granular profiles in the lysosomal storage, as well as normal excretion of subunit c in urine samples, was found in five cases. When the palmitoyle-protein thioesterase (PPT) was studied in these five cases, it was found that the level was deficient, suggesting that they are not LINCL, but the infantile form of neuronal ceroid lipofuscinosis (INCL). Using molecular genetic techniques in the typical JNCL cases, common 1.02 kb deletion to CLN3 was found in 23/27 (homozygotes) and in one allele 4/27 (heterozygotes) in affected pedigrees. In atypical JNCL pedigrees, it was found in 5/16 heterozygotes, while in 1/5 pedigrees, a novel mutation of one atypical JNCL where a single amino acid substitution at 295 E-->K was found in one allele. None of the atypical JNCL cases was homozygote. In atypical JNCL cases where mutation in CLN3 has not been identified (11/16 probands), several possibilities may exist. The phenotype may be caused by a yet undefined mutation in CLN3 or may be due to phenotypically overlapping with other forms of NCL. Pheno/genotypic correlation and the diagnostic difficulties are discussed.

Combined acute necrotic myelopathy (ANM) and cerebellar degeneration associated with malignant disease.

Clinicopathologic findings are reported in the case of an unusual combined degeneration of the cerebellum and spinal cord associated with bronchial carcinoma. Clinical features were sudden sensory onset, rapid and complete flaccid paraplegia with ascending course, sphincter paralysis, rapid impairment of general condition, and high protein content in CSF without inflammatory cell reaction. The neuropathologic findings included massive transverse necrosis at the thoracic level and degeneration of the ascending and descending tracts. The necrotic areas involved mostly white matter without any vascular topography. There was no inflammation or specific vascular alteration. In the cerebellum widespread cortical atrophy was observed. There were no metastases in the cord, meninges, vertebral column, or nerve roots. The simultaneous occurrence of two distinct types of nervous tissue alterations reflects the pathomechanisms effective in CNS reactions to extraneuronal malignancy.

Subacute sensory neuronopathy in small cell cancer of the lung. Immunocytochemical study of 2 cases.

Two cases of subacute sensory neuronopathy (SSN) associated with small cell lung cancer are reported. In both cases motor disability, attributed to motor neuropathy and neuronopathy, respectively, accompanied SSN. Immunohistochemical studies performed provide further data suggesting the involvement of humoral antibodies and the participation of protease inhibitors in the pathogenesis of SSN.

[Diagnostic difficulties in a case of vasogenic lateral pontine syndrome]. / Trudnosci diagnostyczne w przypadku naczyniopochodnego zespolu bocznego mostu.

A 54-year-old male patient is reported in whom progression of clinical manifestations, neurological signs and results of ancillary investigations suggested a cerebellopontine angle tumour in the initial stage of the disease. Further observation and additional appearance after 10 months of signs evidencing damage to other brain-stem structures made possible the diagnosis of a vascular aetiology of the pathological process.

[Hereditary hypersensitivity of the peripheral nerves to pressure]. / Dziedziczna nadwraåliwosc nerwów obwodowych na ucisk.

The authors describe a case of hereditary neuropathy with increased sensitivity to pressure stressing its characteristic course facilitating the diagnosis. Electrophysiological investigations of the peripheral nerves demonstrated a generalized character of the disease process, morphological examination of the peripheral nerves demonstrated presence of characteristic although non-specific, limited myelin thickenings, different length of internodal segments, slight evidence of demyelination and remyelination, and presence of axonal component. The mechanism of development of these changes is still unexplained, possible myelinogenesis disturbances are discussed.