Intracellular trafficking of lysosomal proteins and lysosomes.

In the synthesis and trafficking of precursors of most lysosomal matrix proteins, the stages necessary for lysosomal delivery include the addition of phosphorylated mannose-rich oligosaccharides, binding of the modified proteins to receptors, their segregation from the secretory pathways and delivery to the endosomal pathway. Targeting of both internally synthesized and externally provided enzymes (as in enzyme replacement therapy) to endosomes is executed by a complex machinery of membrane and cytosolic proteins. Recently, the homotypic fusion and vacuolar protein sorting (HOPS) complex has been identified in lysosomes from human cells. This complex is likely to play an important role in the exchange of enzymes between endosomal and lysosomal compartments. The present review describes the interactions and functions of proteins that participate in delivering lysosomal proteins to different lysosomal compartments. In summary, lysosomal trafficking depends on the recognition of many structural signals. It delivers soluble and membrane proteins, and can be exploited for therapeutic substitution of missing enzymes.

Traumatic brain injury as a risk factor for Alzheimer disease. Comparison of two retrospective autopsy cohorts with evaluation of ApoE genotype.

BACKGROUND AND PURPOSE: The impact of traumatic brain injury (TBI) on the pathogenesis of Alzheimer disease (AD) is still controversial. The aim of our retrospective autopsy study was to assess the impact of TBE and ApoE allele frequency on the development of AD. MATERIAL AND METHODS: We examined 1. the incidence of AD pathology (Braak stageing, CERAD, NIA-Reagan Institute criteria) in 58 consecutive patients (mean age +/- SD 77.0 +/- 6.8 years) with residual closed TBI lesions, and 2. the frequency of TBI residuals in 57 age-matched autopsy proven AD cases. In both series, ApoE was evaluated from archival paraffin-embedded brain material. RESULTS: 1. TBE series: 12.1 % showed definite and 10.3% probable AD (mean age 77.6 and 75.2 years), only 2/13 with ApoEepsilon3/4. From 45 (77.6%) non-AD cases (mean age 78.2 years), 3 had ApoEepsilon3/4. The prevalence of 22.4% AD in this small autopsy cohort was significantly higher than 3.3% in a recent large clinical series and 14% in the general population over age 70. 2. In the AD cohort with ApoEepsilon4 allele frequency of 30% similar to other AD series, residuals of closed TBI were seen in 4 brains (7%) (mean age +/- SD 78.2 +/- 6.4), all lacking the ApoEepsilon4 allele. TBI incidence was slightly lower than 8.5% in the clinical MIRAGE study. CONCLUSIONS: The results of this first retrospective autopsy study of TBI, ApoEepsilon allele frequency, and AD confirm clinical studies suggesting severe TBI to be a risk factor for the development AD higher in subjects lacking ApoEepsilon4 alleles. Further studies in larger autopsy series are needed to elucidate the relationship between TBI, genetic predisposition, and AD.

CDKN2A/p16 inactivation is related to pituitary adenoma type and size.

p16 (CDKN2A, MTS1, INK4A) status at genomic and protein levels was analysed and correlated with clinico-pathological features in 72 pituitary adenomas. Methylation of CpG islands of promoter/exon 1 sequences was found in most gonadotroph, lactotroph, plurihormonal, and null cell adenomas (36 of 44, 82%), but it was rare in somatotroph (1 of 13 cases, 8%) and corticotroph adenomas (1 of 15 cases, 7%). Homozygous CDKN2A deletion was restricted to rare somatotroph (15%) and corticotroph adenomas (13%). Immunohistochemical p16 protein expression was observed in the normal adenohypophysis, whereas it was absent in 60 of 72 (83%) tumours and reduced in another ten (14%) tumours. Staining for p16 was only seen in 5 of 15 (33%) corticotroph, 3 of 13 (23%) somatotroph, 3 of 5 (60%) plurihormonal, and 1 of 19 (5%) null cell adenomas. p16 immunonegativity without CDKN2A methylation or deletion occurred in 22 tumours, including most somatotroph and corticotroph adenomas (15 of 28, 54%). Both CDKN2A alterations and p16 negativity were related to larger tumour size. Patients with p16-negative tumours were older than patients with p16-positive tumours. These data suggest that p16 down-regulation is common in all adenoma types. The mechanisms of p16 down-regulation probably involve CDKN2A methylation in most types, but remain to be determined in somatotroph and corticotroph adenomas. These findings also suggest that p16 down-regulation is usually not an initial event, but is acquired during adenoma progression.

Effects of closed traumatic brain injury and genetic factors on the development of Alzheimer's disease.

In order to assess the impact of traumatic brain injury (TBI) and Apolipoprotein E (ApoE) allele frequency on the development of Alzheimer's disease (AD), we examined: (i) the incidence of AD pathology in 55 consecutive autopsy cases (mean age +/- SD 77.6 +/- 7.3 years) with residual closed TBI lesions and (ii) the frequency of TBI residuals in 53 age-matched autopsy proven AD cases. In both series, ApoE was evaluated from archival paraffin-embedded brain material. The results were as follows: (i) In the TBI series, 12.7% showed Consortium to Establish a Registry for Alzheimer's disease (CERAD) definite and 9.1% probable AD, only one with ApoEepsilon4. From the remaining 43 non-AD cases, three had ApoEepsilon4. The prevalence of 21.8% AD in this small autopsy cohort was significantly higher than 3.3% in a recent large clinical series and 14% in the general population over the age of 70. (ii) In the AD cohort with ApoEepsilon4 allele frequency of 30% similar to other AD series, residuals of TBI were seen in 4 brains (7.5%), all lacking the ApoEepsilon4 allele. TBI incidence was slightly higher than 8.5% in the clinical MIRAGE study. The results of this first retrospective autopsy study of TBI, ApoE allele frequency, and AD confirms clinical studies suggesting severe TBI to be a risk factor for the development of AD particularly in subjects lacking ApoEepsilon4.

COL9A2 allelotypes in intervertebral disc disease.

An allelic variation of the COL9A2 gene encoding the alpha(2)-chain of collagen IX has recently been identified as a genetic risk factor for intervertebral disc prolapse, resulting in a tryptophane (Trp) substitution at position 326 of the protein. To enable quick screening of a large population we established a single enzyme (BsmFI) restriction assay which was validated by screening disc tissue samples of 250 patients (age, 47.1 +/- 13.7 years). Positive results were confirmed by nucleotide sequencing. The Trp allele was found in three patients (1.2%) who suffered from their first prolapse and were significantly older (70.7 +/- 8.5 years) than the other 247 patients. Since the substitution affects a domain covalently linked to collagen II fibrils, we conclude that this allelotype may contribute to reduced collagen crosslinking, disc instability and eventually prolapse in the elderly.

Diagnosis of tachycardia syndromes associated with orthostatic symptoms.

The postural tachycardia syndrome (POTS) is characterized by excessive tachycardia only in upright position without evidence of a cardiac or metabolic disease in combination with orthostatic symptoms like dizziness, lightheadedness or syncope but without relevant falls in blood pressure. The cause is unknown. A specific diagnostic marker has not been found so far. Eighteen patients with typical symptoms of POTS were examined. They underwent standard autonomic function tests with continuous measurement of heart rate (HR) and blood pressure. All fulfilled the inclusion criteria of pathologically increased HR activation during passive tilt or standing over 90 seconds. The upper limits of normal were based on data from 137 healthy volunteers between 18 and 85 years of age. Actively standing up induced more POTS-typical HR increases and lead to more consistent results than passive tilt. HR responses during Valsalva manoeuvre and deep breathing were normal in all except one patient each, indicating that assessment of HR during these tests does not contribute to the diagnosis of POTS. Frequency of symptoms reducing overall well-being and the degree of impairment of life quality by symptoms typical of POTS were measured with a self-assessment scale. The majority of patients reported a permanent reduction of overall well-being and a relevant impairment of life quality due to dizziness, tachycardia, and syncopes. This underlines the importance of considering POTS as a differential diagnosis of orthostatic syndromes and the necessity of treating it adequately.

Proteoglycan form of colony-stimulating factor-1 (proteoglycan-100). Stimulation of activity by glycosaminoglycan removal and proteolytic processing.

A proteoglycan had been isolated from the conditioned media of a human osteosarcoma cell line and had tentatively been named proteoglycan-100 (PG-100) because of the size of its core glycoprotein. Amino acid sequencing of the purified proteoglycan and cDNA analysis were consistent with the assumption that PG-100 is identical with the proteoglycan form of CSF-1 (or macrophage colony-stimulating factor). PG-100 induced mouse macrophage differentiation. Proliferation of macrophages was stimulated in a dose-dependent manner. On a molar basis, however, about 100- to 300-fold higher doses of PG-100 than of recombinant human (rh)CSF-1 were required for the half-maximal growth-stimulating effect. Upon enzymatic removal of the glycosaminoglycan chain, the purified core protein exhibited higher activity, but was still about 20-fold less active than rhCSF-1. Incubation of the purified proteoglycan for 48 h at 37 degrees C led to the formation of a glycosaminoglycan-free 50-kDa fragment either by autoproteolysis or by the action of a protease not yet identified. The purified fragment exhibited almost the same biologic activity as rhCSF-1. The glycosaminoglycan chain of the growth factor was not only shown to inhibit CSF-1 activity but also to increase the stability of the core protein when the CSF-1-producing osteosarcoma cells were maintained in a collagen lattice. These findings provide a link between a soluble, highly active cytokine and its extracellular matrix storage form of comparatively low activity.