The monoclonal antibody, Tau-1, which had previously been used to localize tau to the axonal compartment in brain has been reutilized for light and electron microscopic immunohistochemistry following phosphatase treatment of tissue. We report here that a significant quantity of tau in the central nervous system is phosphorylated in situ at or near the Tau-1 epitope, preventing the binding of the Tau-1 antibody. Upon removal of this/these phosphate group(s), however, Tau-1 was observed in the somatodendritic compartment of neurons as well as in axons. Furthermore, intense staining was also observed in astrocytes and in perineuronal glial cells. This immunoreactivity was present along the lengths of microtubules and on ribosomes (polysomes). Treatment of immunoblots of extracts of whole cerebral cortex with phosphatase confirmed the immunohistochemical results in that a 50-65% increase in Tau-1 binding to the tau region of the blot was noted. Moreover, a novel monoclonal antibody, Tau-2, was also used in these experiments. This antibody binds only to tau and localizes along microtubules in axons, somata, dendrites, and astrocytes and on ribosomes (polysomes) without phosphatase pretreatment.

We present evidence that the microtubuleassociated protein tau is present in oligodendrocytes (OLGs), the central nervous system cells that make myelin. By showing that tau is distributed in a pattern similar to that of myelin basic protein, our results suggest a possible involvement of tau in some aspect of myelination. Tau protein has been identified in OLGs in situ and in vitro. In interfascicular OLGs, tau localization, revealed by monoclonal antibody Tau-5, was confined to the cell somata. However, in cultured ovine OLGs with an exuberant network of processes, tau was detected in cell somata, cellular processes, and membrane expansions at the tips of these processes. Moreover, in such cultures, tau appeared localized adjacent to or coincident with myelin basic protein in membrane expansions along and at the ends of the cellular processes. The presence of tau mRNA was documented using fluorescence in situ hybridization. The distribution of the tau mRNA was similar to that of the tau protein.Western blot analysis of cultured OLGs showed the presence of many tau isoforms. Together, these results demonstrate that tau is a genuine oligodendrocyte protein and pave the way for determining its functional role in these cells.

Neurofilament light chain polypeptide (NEFL) is one of the most abundant cytoskeletal components of the neuron. Mutations in the NEFL gene were recently reported as a cause for autosomal dominant Charcot-Marie-Tooth type 2E (CMT2E) linked to chromosome 8p21. In order to investigate the frequency and phenotypic consequences of NEFL mutations, we screened 323 patients with CMT or related peripheral neuropathies. We detected six disease associated missense mutations and one 3-bp in-frame deletion clustered in functionally defined domains of the NEFL protein. Patients have an early onset and often a severe clinical phenotype. Electrophysiological examination shows moderately to severely slowed nerve conduction velocities. We report the first nerve biopsy of a CMT patient with a de novo missense mutation in NEFL, and found an axonal pathology with axonal regeneration clusters and onion bulb formations. Our findings provide further evidence that the clinical variation observed in CMT depends on the gene mutated and the specific type of mutation, and we also suggest that NEFL mutations need to be considered in the molecular evaluation of patients with sporadic or dominantly inherited CMT.

Mutations of the ganglioside-induced differentiation-associated protein 1 gene (GDAP1) cause autosomal recessive Charcot-Marie-Tooth disease type 4A. We report four additional families with recessive mutations (487C-->T, Q163X; 359G-->A, R120Q) of GDAP1; Q163X occurred in three unrelated Hispanic families that had the same haplotype suggesting a Spanish founder mutation. Both the Q163X and the R120Q mutation cause demyelination and axonal loss. The patients had symptoms within the first two years of life and involvement of cranial, sensory, and enteric nerves. Neuropathology showed loss of large myelinated fibers, onion bulb formations and focal folding of the outer myelin lamina.

We have shown previously that glycogen synthase kinase-3␤ (GSK-3␤), cyclin-dependent kinase 5, and c-Jun NH2-terminal kinase become overactivated and hyperphosphorylate in heat-shocked female rats. This hyperphosphorylation of is estrogen-independent, prevented by androgens, and similar to Alzheimer's disease. In this study, ovariectomized (OVX) Sprague-Dawley rats (n ‫؍‬ 75) received daily injections of 10 g of 17␤-estradiol benzoate (EB), or 250 g of testosterone propionate (TP), or both EB and TP, or sesame oil (SO) vehicle for 4 -6 weeks. In kinase assays of forebrain homogenates, overactivation of GSK-3␤ at 0 -6 h after heat shock toward human recombinant , bovine , and phosphoglycogen synthase peptide 2 was prevented in OVX ؉ TP and OVX ؉ (EB ؉ TP) but not in sham-OVX ؉ SO, OVX ؉ SO, and OVX ؉ EB. Abs against inactive (pSer 9 ) and activity-enhanced (pTyr 216 ) GSK-3␤ showed marked increase of pSer 9 -and decrease of pTyr 216 -GSK-3␤ in both OVX ؉ TP and OVX ؉ (EB ؉ TP) but not in sham-OVX ؉ SO, OVX ؉ SO, and OVX ؉ EB. EB enhanced the overactivation of cyclin-dependent kinase 5. The activity of c-Jun NH2-terminal kinase was gonadal hormone-independent. The serum concentrations of testosterone and 17␤-estradiol were 2.53 ng͞ml and 201 pg͞ml in OVX ؉ TP and OVX ؉ EB, respectively. These findings demonstrate that testosterone prevents the hyperphosphorylation of by inhibiting the heat shock-induced overactivation of GSK-3␤ and suggest that androgens given to aging men or, in combination with estrogens, to postmenopausal women could prevent or delay Alzheimer's disease.A bout two times more women than men have Alzheimer's disease (AD) (1), partly because women with AD live longer. However, recent studies showed that women carry an innate higher risk for AD (2). The precipitous decline and loss of neuroprotective effects of estrogens in postmenopausal women-in contrast to the gradual decline of androgens in aging men-are offered as an explanation. However, recent studies showed no beneficial effects of estrogens on mild-to-moderate AD (3). On the other hand, a possible advantageous role of androgens in the prevention and͞or treatment of AD has not been tried yet despite their neuroprotective effects (4), the century-old suggestion that they may rejuvenate aged men (5), lower serum testosterone concentration in men with AD (6), the increasing evidence that stressful stimuli play a role in the etiopathogenesis of AD (7,8), and inhibition of stress response by androgens (9).The cause of AD is not known, but it seems to be a syndrome resulting from an interplay among a genetic predisposition, environmental stress factors, and the aging process. The histologic hallmarks of AD are the senile plaques made of A␤ amyloid, dystrophic neurites, and reactive glial cells, and the neurofibrillary tangles composed of bundles of abnormal filaments, the so-called paired-helical filaments, the major component of which is hyperphosphorylated (10-12). However, the earliest manifestation of hyperphosphorylated is a granular form in...

ABSTRACT8,,8'-Iminodipropionitrile (IDPN), a synthetic compound that selectively impairs slow axonal transport, produced a rearrangement of the axonal cytoskeleton, smooth endoplasmic reticulum, and mitochondria . Immunoperoxidase staining using an antiserum to the 68,000-dalton neurofilament subunit demonstrated a displacement of neurofilaments toward the periphery of the axons of IDPN-treated rats . This change occurred simultaneously along the entire length of the sciatic nerve . Ultrastructural morphometry of the axonal organelles confirmed the peripheral relocation of neurofilaments and also showed a displacement of microtubules, smooth endoplasmic reticulum, and mitochondria to the center of the axons. The overall density of axonal mitochondria was increased, whereas those of other organelles were not significantly changed. Axons were reduced in size by 10-24%, the large axons being more affected than the small ones . The observed rearrangement of axonal organelles may be due to an effect of IDPN on microtubule-neurofilament interactions, which could in turn explain the impairment of the slow transport. Axons in IDPN intoxication are a useful model to study the organization of the axoplasm and the mechanism of axonal transport.

We have shown that heat shock induces rapid dephosphorylation of in both female and male rats followed by hyperphosphorylation only in female rats. To investigate the role of gonadal hormones, rats were ovariectomized (OVX), orchiectomized (ORX), or sham-gonadectomized and received replacement therapy with estradiol benzoate (EB), testosterone propionate (TP), or sesame oil (SO) vehicle for 2-3 weeks, respectively. At 0, 3, 6, and 12 hr after heat shock, immunoblot analysis of SDS cerebral extracts was performed using phosphate-dependent and -independent anti-antibodies. Seven groups of rats were analyzed: (i) sham-OVX ؉ SO; (ii) OVX ؉ SO; (iii) OVX ؉ EB; (iv) sham-ORX ؉ SO; (v) ORX ؉ SO; (vi) ORX ؉ TP; and (vii) ORX. In all seven groups, there was dephosphorylation of at 0 hr after heat shock. In all three groups of female rats, there was hyperphosphorylation of at 3 hr after heat shock, and its degree and temporal pattern were identical between the OVX ؉ SO and OVX ؉ EB groups. In male rats, there was hyperphosphorylation of at 3 hr after heat shock in both ORX ؉ SO and ORX groups, and its degree was reduced in the ORX ؉ TP group. Thus, dephosphorylation of is gonadal hormoneindependent, but while its hyperphosphorylation is estrogenindependent it is prevented by androgens. Because is abnormally hyperphosphorylated in Alzheimer disease, which is more frequent in women than men, these findings suggest that androgens may exert a neuroprotective effect.The prevalence of Alzheimer disease (AD) after age 65 is roughly two to three times higher in women than men (1). Because estrogens prevent neuronal atrophy (2), exert a neuroprotective effect against various toxic insults (3), and may synergize with neurotrophic factors (4-6), a deficiency of estrogens has been suggested to play a role in the pathogenesis of AD. However, estrogen neurotoxicity also has been postulated to be involved in brain aging and degeneration (7). So far, in many studies of postmenopausal women, demonstration of a beneficial effect of estrogen replacement therapy in reducing the risk or delaying the onset of AD has been controversial (8, 9). On the other hand, a possible advantageous role of androgens in reducing AD occurrence or severity has not been investigated yet. This is in spite of the century-old suggestion that androgens may rejuvenate aged men (10), the demonstration that androgens inhibit the stress response (11-13), and the increasing evidence that stressful stimuli play a role in the etiopathogenesis of AD (14-16).One histologic hallmark of AD is the neurofibrillary tangle. Neurofibrillary tangles are perikaryal aggregates of abnormal filaments, the so-called paired helical filaments, which consist mostly of abnormally hyperphosphorylated (17,18), and appear to be conjugated with the stress-inducible protein ubiquitin (16).is a group of Ϸ50-66-kDa phosphopolypeptides, but although many protein kinases and phosphatases have been described, the lack of appropriate animal models hindered so far the elucidation of the cascade...

We have examined the distribution of microtubule-associated protein 2 (MAP2) in the lumbar segment of spinal cord, ventral and dorsal roots, and dorsal root ganglia of control and beta,beta'-iminodipropionitrile- treated rats. The peroxidase-antiperoxidase technique was used for light and electron microscopic immunohistochemical studies with two monoclonal antibodies directed against different epitopes of Chinese hamster brain MAP2, designated AP9 and AP13. MAP2 immunoreactivity was present in axons of spinal motor neurons, but was not detected in axons of white matter tracts of spinal cord and in the majority of axons of the dorsal root. A gradient of staining intensity among dendrites, cell bodies, and axons of spinal motor neurons was present, with dendrites staining most intensely and axons the least. While dendrites and cell bodies of all neurons in the spinal cord were intensely positive, neurons of the dorsal root ganglia were variably stained. The axons of labeled dorsal root ganglion cells were intensely labeled up to their bifurcation; beyond this point, while only occasional central processes in dorsal roots were weakly stained, the majority of peripheral processes in spinal nerves were positive. beta,beta'- Iminodipropionitrile produced segregation of microtubules and membranous organelles from neurofilaments in the peripheral nervous system portion and accumulation of neurofilaments in the central nervous system portion of spinal motor axons. While both anti-MAP2 hybridoma antibodies co-localized with microtubules in the central nervous system portion, only one co-localized with microtubules in the peripheral nervous system portion of spinal motor axons, while the other antibody co-localized with neurofilaments and did not stain the central region of the axon which contained microtubules. These findings suggest that (a) MAP2 is present in axons of spinal motor neurons, albeit in a lower concentration or in a different form than is present in dendrites, and (b) the MAP2 in axons interacts with both microtubules and neurofilaments.