Accumulating evidence suggests that mitochondrial dysfunction and oxidative stress play major roles in aging. Chronic administration of D-galactose has been reported to cause deterioration of cognitive and motor skills that are similar to symptoms of aging and, therefore, is regarded as a model of accelerated aging. Because enhancing endogenous antioxidants is now widely regarded as an attractive therapy for conditions associated with mitochondrial oxidative stress, in the present study the effects of a-lipoic acid, L-carnitine, and PMX-500F on D-galactose treated mice were tested. Female mice were injected with (100 mg/kg) D-(þ)-galactose for 6 weeks and some groups were treated with a daily dose of a-lipoic acid (5 mg/kg), L-carnitine (3.9 mg/kg), PMX-500F (11.9 mg/kg), or the vehicle (0.1 M Tris, pH 7.4). Control mice were treated with physiological saline. An accelerating Rota-Rod, open field test, and Y-maze test were performed, and serum lactate concentrations were analyzed. These analyses did not identify impairment in motor coordination, open-field activity, or spatial memory ( p > 0.05). Similarly, serum lactate concentrations in D-galactose-treated mice were not elevated when compared to controls ( p > 0.05). Treatment with the antioxidant compounds at the given concentrations did not result in any changes in the behavioral parameters tested. In conclusion, results of this study illustrate that chronic, short-term D-galactose treatment may not represent a suitable model for inducing readily detectable age-related neurobehavioral symptoms in mice.

Reactive astrocytes form a scar after injury to the CNS that many investigators believe contributes to the lack of functional regeneration. In the present study, we identify an astrocytic membrane protein that appears to play an important role in reactive gliosis and scar formation. Cultures of rat astrocytes were used as a model system to produce and to screen monoclonal antibodies that would alter cell growth. One antibody, AMP1, was identified that depresses the mitotic activity of cultured glial cells and alters their morphology. Expression cloning reveals that the antigen on the external surface of the cultured glial cells has a high degree of homology with the human lymphocyte protein called Target of the Anti-Proliferative Antibody (TAPA-1; this rat protein will be referred to as rTAPA). rTAPA is a member of the tetramembrane-spanning superfamily of proteins and, as with other members of this family of proteins, rTAPA is associated with the regulation of cellular interactions and mitotic activity. After an injury to the cerebral cortex, there is a dramatic increase in AMP1 immunoreactivity that is spatially restricted to the reactive astrocytes at the glial scar. This change represents an upregulation of a membrane protein, rTAPA, that is approximately equal to the increase observed for glial fibrillary acidic protein. The high levels of rTAPA at the site of CNS injury and the AMP1 antibody perturbation studies indicate that rTAPA may play a prominent role in the response of astrocytes to injury and in glial scar formation.

Abstract. To examine the ultrastructural distribution of laminin within kidney basement membranes, we prepared rat anti-mouse laminin mAbs to use in immunolocalization experiments. Epitope domains for these mAbs were established by immunoprecipitation, immunoblotting, affinity chromatography, and rotary shadow EM. One mAb bound to the laminin A and B chains on blots and was located to a site •15 nm from the long arm-terminal globular domain as shown by rotary shadowing. Conjugates of this long arm-specific mAb were coupled to horseradish peroxidase (HRP) and intravenously injected into mice. Kidney cortices were fixed for microscopy 3 h after injection. HRP reaction product was localized irregularly within the renal glomerular basement membrane (GBM) and throughout mesangial matrices. In addition, this rnAb bound in linear patterns specifically to the laminae rarae of basement membranes of Bowman's capsule and proximal tubule. This indicates the presence of the long arm immediately beneath epithelial cells in these sites. The laminae densae of these basement membranes were negative by this protocol. In contrast, the lamina ram and densa of distal tubular basement membranes (TBM) were both heavily labeled with this mAb. A different ultrastructural binding pattern was seen with eight other mAbs, including two that mapped to different situs on the short arms by rotary shadowing and five that blotted to a large pepsinresistant laminin fragment (P1). These latter mAbs bound weakly or not at all to GBM but all bound throughout mesangial matrices. In contrast, discrete spots of HRP reaction product were seen across all layers of Bowman's capsule BM and proximal TBM. These same mAbs, however, bound densely across the full width of distal TBM. Our findings therefore show that separate strata of different basement membranes are variably immunoreactive to these laminin mAbs. The molecular orientation or integration of laminin into the three dimensional BM meshwork therefore varies with location. Alternatively, there may be a family of distinct laminin-like molecules distributed within basement membranes.

Preclinical Research In this review, we discuss epigenetic-driven methods for treating neurodegenerative disorders associated with mitochondrial dysfunction, focusing on carnitinoid antioxidant-histone deacetylase inhibitors that show an ability to reinvigorate synaptic plasticity and protect against neuromotor decline in vivo. Aging remains a major risk factor in patients who progress to dementia, a clinical syndrome typified by decreased mental capacity, including impairments in memory, language skills, and executive function. Energy metabolism and mitochondrial dysfunction are viewed as determinants in the aging process that may afford therapeutic targets for a host of disease conditions, the brain being primary in such thinking. Mitochondrial dysfunction is a core feature in the pathophysiology of both Alzheimer and Parkinson diseases and rare mitochondrial diseases. The potential of new therapies in this area extends to glaucoma and other ophthalmic disorders, migraine, Creutzfeldt-Jakob disease, post-traumatic stress disorder, systemic exertion intolerance disease, and chemotherapy-induced cognitive impairment. An emerging and hopefully more promising approach to addressing these hard-to-treat diseases leverages their sensitivity to activation of master regulators of antioxidant and cytoprotective genes, antioxidant response elements, and mitophagy. Drug Dev Res 77 : 109-123, 2016. © 2016 Wiley Periodicals, Inc.

Neuropsychiatric disorders are a heterogeneous group of conditions that often share underlying mitochondrial dysfunction and biological pathways implicated in their pathogenesis, progression, and treatment. To date, these disorders have proven notoriously resistant to molecular-targeted therapies, and clinical options are relegated to interventional types, which do not address the core symptoms of the disease. In this review, we discuss emerging epigenetic-driven approaches using novel acylcarnitine esters (carnitinoids) that act on master regulators of antioxidant and cytoprotective genes and mitophagic pathways. These carnitinoids are actively transported, mitochondria-localizing, biomimetic coenzyme A surrogates of short-chain fatty acids, which inhibit histone deacetylase and may reinvigorate synaptic plasticity and protect against neuronal damage. We outline these neuroprotective effects in the context of treatment of neuropsychiatric disorders such as autism spectrum disorder and schizophrenia.

A series of monoclonal antibodies was prepared against the pepsin-resistant fragment of type IX collagen designated HMW. One of these antibodies (called 2C2) was selected for further analysis. Antibody 2C2 showed no cross-reactivity with other collagen types by inhibition enzyme-linked immunosorbent assays. It recognized an epitope present in native HMW, but failed to recognize any of the three chains of HMW fractionated after denaturation followed by reduction and alkylation of interchain disulfide bridges. Electron microscopic observations after rotary shadowing showed that the location of the epitope for antibody 2C2 was close to the carboxy-terminus of HMW. Immunofluorescent staining of sections of embryonic and adult cartilage with antibody 2C2 after removal of proteoglycans by testicular hyaluronidase digestion showed that type IX collagen is distributed throughout the cartilage matrix, and is not present in other connective tissues or skeletal muscle. The intact type IX collagen molecule, which was secreted by a suspension culture of freshly isolated embryonic chick chondrocytes, was recognized by rotary shadowing in the presence of antibody 2C2 after first precipitating the procollagens from the culture medium with ammonium sulfate (30%). Two different collagenous molecules were present in the precipitate: a longer molecule of type II procollagen (average length, 335 nm) with both amino-and carboxy-propeptides still remaining uncleaved, and a shorter molecule (average length, 190 nm) which was identified as type IX collagen. Antibody 2C2 consistently bound to the shorter molecules at a site located 136 nm from a distinctive knob at one end of the molecule, and did not bind to any specific site on the type II procollagen molecules. The structure of the intact type IX collagen molecule with the location of both collagenous and noncollagenous domains was as predicted after converting the nucleotide sequence of a cDNA clone encoding for one of the chains of type IX collagen to an amino acid sequence (Ninomiya, Y., and B. R. Olsen, 1984, Proc. Natl. Acad. Sci. USA., 81:3014-3018).Type IX collagen was initially isolated from chicken sternum as two pepsin-resistant fragments designated HMW and LMW (1, 2) or MI and M2 (3). Subsequent studies showed that the intact molecule from which these fragments are derived is much larger (4-7), and that both fragments may be derived from a single molecule (6). Three genetically distinct chains were fractionated from both LMW (8, 9) and HMW (10). The three chains were consistently present in a l:l:l proportion for both HMW and LMW, and it was proposed that there is a single type IX molecule of chain organization 814 al(IX)a2(lX)a3(lX) (8-10). A cDNA clone (called pYN 1738) was prepared from chick chondrocyte RNA and shown to encode for one of the chains of both HMW and LMW (11). The DNA sequence of this clone was converted to an amino acid sequence and this sequence compared to the amino acid compositions of the tryptic peptides and the amino-terminal sequences of t...

We studied the interactions between human neutrophils, as well as the purified human neutrophil serine proteases elastase (HNE) and cathepsin G (HNCG), and laminin. Our results show that intact laminin and two proteolytic fragments generated by HNE bind to neutrophils and stimulate cell migration. Domain-specific antilaminin monoclonal antibodies, rotary shadowing electron microscopy, and Western blotting mapped the two promigratory fragments on the laminin cross to the apical three-armed region and long arm, respectively. In contrast, a fragment derived from the terminal ends of short arms neither bound to neutrophils nor stimulated migration. When neutrophils embedded in a reconstituted basement membrane gel were activated with phorbol myristate acetate, several stable, proteolytic laminin fragments were released into supernatants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting showed that these fragments appeared identical to those generated after digestion of soluble laminin with HNE and HNCG. Furthermore, release of laminin fragments by embedded neutrophils was inhibited by diisopropyl fluorophosphate, and duplicated by incubating the basement membrane gel with purified HNE and HNCG. Our findings therefore suggest that neutrophils, through release of HNE and HNCG, are capable of digesting basement membrane laminin in vivo. In addition, the release of laminin fragments from damaged basement membranes may promote neutrophil migration and thereby accelerate inflammatory processes.

The location of the epitopes for monoclonal antibodies against chicken type IV and type V collagens were directly determined in the electron microscope after rotary shadowing of antibody/collagen mixtures . Three monoclonal antibodies against type IV collagen were examined, each one of which was previously demonstrated to be specific for only one of the three pepsin-resistant fragments of the molecule . The three native fragments were designated (F1)2 F2, F3, and 7S, and the antibodies that specifically recognize each fragment were called, respectively, IA8, 111312, and ID2 . By electron microscopy, monoclonal antibody IA8 recognized an epitope located in the center of fragment (F1) 2 F2 and in tetramers of type IV Collagen at a distance of 288 nm from the 7S domain, the region of overlap of four type IV molecules . Monoclonal antibody 111312, in contrast, recognized an epitope located only 73 nm from the 7S domain . This result therefore provides direct visual evidence that the F3 fragment is located closest to the 7S domain and the order of the fragments must be 7S-F3-(Fl)2 F2 . The epitope for antibody ID2 was located in the overlap region of the 7S domain, and often several antibody molecules were observed binding to a single 7S domain . The high frequency with which antibody molecules were observed to bind to fragments of type IV collagen suggests that there is a single population of type IV molecules of chain organization [al(IV)] 2 a2(IV), and that four identical molecules must form a tetramer that is joined in an antiparallel manner at the 7S domain .The monoclonal antibodies against type V Collagen, called AB12 and DH2, were both found to recognize epitopes close to one another, the epitopes being located 45-48 nm from one end of the type V collagen molecule. The significance of this result still remains uncertain, but suggests that this site is probably highly immunoreactive . It may also be related to the specific cleavage site of type V collagen by selected metalloproteinases and by a-thrombin. This cleavage site is also known to be located close to one end of the type V molecule .Monoclonal antibodies of high affinity prepared against the different types of collagen potentially have several advantages when compared to conventionally prepared polyclonal antibodies (reviewed in reference 1) . Several groups have now described the preparation of monoclonal antibodies of high specificity for each of the five major collagen types (2-9) . In THE JOURNAL OF CELL BIOLOGY " VOLUME 98 MAY 1984 1637-1644 C The Rockefeller University Press -0021-9525/84/05/1637/08 $1 .00 previous experiments, we have prepared monoclonal antibodies that specifically recognized epitopes present in native molecules of chicken type IV collagen (10, 11) and type V collagen (12). These antibodies are now being used as reagents to investigate the macromolecular organization and function ofthese collagens in several tissues including lens capsule (13),