Compromised mitochondrial function leads to increased cytosolic calcium and to activation of MAP kinases.

We have investigated in rat pheochromacytoma PC12 cells the activation of the mitogen-activated protein kinases ERK1 and ERK2 by the mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP). This treatment slowly decreases ATP levels to 30% of control, whereas the internal calcium level rises very rapidly to 250% of control, derived from internal stores. Tyrosine phosphorylation of ERK1 and ERK2 increases gradually, starting after 5 min of treatment, to reach a maximum at 30 min; the kinase activity reaches 250% when measured after 1 hr of treatment. The drop in ATP levels is slower still. Comparison of the time courses of the rapid rise in cytosolic calcium with the slower increase in ERK1 and ERK2 activation suggests one or more intermediate stages in this pathway. Chelation of cytosolic calcium with dimethyl bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid abolished the FCCP-stimulated rise in internal calcium, as well as the tyrosine phosphorylation and the activation of the ERKs. Surprisingly, caffeine, which releases calcium from different internal stores, did not increase the tyrosine phosphorylation and did not activate the ERKs. The FCCP effect on calcium storage may be related to mitochondrial dysfunction in Alzheimer disease, which might result in ineffective buffering of cytosolic calcium that leads to mitogen-activated protein kinase activation and subsequent protein phosphorylations.

Aggregates of a beta-amyloid peptide are required to induce calcium currents in neuron-like human teratocarcinoma cells: relation to Alzheimer's disease.

We report that human hNT cells display neuron-like calcium channel activation. Patch-clamp experiments show that exposure of hNT cells to the Alzheimer-related amyloid peptide beta AP(25-35) induces large and irreversible inward calcium currents at -80 mV in whole cell mode, with a linear current-voltage relationship. This behavior is suggestive of ionophore formation. An analogous peptide with scrambled sequence has no effect. These ionophore effects by the beta AP(25-35) peptide, the first report in a human cell-line, are very rapid effects. The currents are large and stable, and are blocked by Al3+ but not by Cd2+. Filtration removes a peptide aggregate from the amyloid peptide beta AP(25-35) solution and thereby abolishes the inward current. The residual soluble peptide has no effect. These data suggest that the initial step of the neurotoxic effect of beta AP(25-35) may be due to the insertion of the aggregated peptide into the cellular membrane as a Ca2(+)-carrying ionophore. The relevance of calcium-mediated cell death, especially in Alzheimer's disease, is discussed.

Mechanism and prevention of neurotoxicity caused by beta-amyloid peptides: relation to Alzheimer's disease.

In Alzheimer's disease, neurotoxic beta-amyloid peptides cause a deleterious influx of calcium ions into neurons. This increase in [Ca2+]int is expected to trigger intracellular events that eventually cause cell dysfunction and cell death. We find that the aggregated beta-amyloid peptide beta AP25-35 opens irreversibly a Ca(2+)-carrying channel, as does aggregated beta AP1-42. The opening of this channel is unaffected by DL-AP5, but it is blocked by Mg2+, CNQX and DNQX, suggesting a non-NMDA channel. External calcium enters and cytosolic calcium levels rise several-fold, as measured by fura-2 ratiometric analysis. Our findings illustrate a very early molecular event in the neurotoxicity of Alzheimer's disease. To combat the neurotoxic effect of aggregated beta-amyloid peptides, we have devised a series of very short antagonistic peptides. Using a combinatorial library of hexapeptides made from D-amino acids, we have selected peptides by their ability to complex with the tagged beta-amyloid peptide beta AP25-35. Certain of these so-called 'decoy peptides', as well as some modified decoy peptides, are able to abolish the calcium influx caused by aggregated, probably fibrillar, beta-amyloid peptides beta AP25-35 and beta AP1-42.

Hyperphosphorylation of the cytoskeletal protein Tau by the MAP-kinase PK40erk2: regulation by prior phosphorylation with cAMP-dependent protein kinase A.

PK40erk2 is a MAP kinase which phosphorylates recombinant hTau40 up to 14 moles of phosphate/mole, markedly slowing its electrophoretic mobility. PK40erk2 acting on TAU is expected to cause the appearance of Alzheimer's disease-specific phosphoepitopes, detectable by specific antibodies. Maximal phosphorylation in vitro of hTau40 by PKAcat incorporates only 2-3 moles of phosphate/mole. Consequent, but smaller, reduction in electrophoretic mobility is seen, but not the formation of Alzheimer-specific or hyperphosphorylation-specific epitopes. Phosphorylation of hTau40 by PKAcat sharply reduces the number of phosphates that can now be introduced by PK40erk2 to 5-6 moles/mole, instead of the expected 11 moles/mole. Thus, prior phosphorylation by PKA, a non-proline-directed protein kinase, regulates the conformation of the protein substrate Tau so as to make some sites very much less accessible to phosphorylation by the proline-directed kinase, PK40erk2.

Gene assignment by polymerase chain reaction: localization of the human potassium channel IsK gene to the Down's syndrome region of chromosome 21q22.1-q22.2.

Gene mapping, using the polymerase chain reaction (PCR) on DNA obtained from a human/rodent hybrid cell line carrying only the human chromosome 21, permitted the assignment of the human IsK gene, encoding a slowly activating potassium channel, to chromosome 21. PCR analysis of two complete panels of human/rodent hybrid DNA mapped IsK to chromosome 21 with 100% concordance. By performing PCR on DNA of a human chromosome 21 regional mapping panel the gene was sublocalized to chromosome 21q22.1-q22.2, which also contains the putative Down's syndrome (trisomy 21) region. The PCR product obtained from the hybrid cell line DNA carrying only human chromosome 21 was sequenced, thus confirming that the PCR product was derived from human IsK.

Activation of a neurofilament kinase, a tau kinase, and a tau phosphatase by decreased ATP levels in nerve growth factor-differentiated PC-12 cells.

Brain pathology in Alzheimer disease and in aged controls shows hyperphosphorylation of tau and of neurofilament proteins. Roder and Ingram [Roder, H.M. & Ingram, V.M. (1991) J. Neurosci. 11, 3325-3343 and Roder, H.M., Eden, P.A. & Ingram, V.M. (1993) Biochem. Biophys. Res. Commun. 193, 639-647] previously reported that the brain protein kinase PK40erk can hyperphosphorylate both tau and neurofilaments and interestingly, is strongly inhibited by ATP uncomplexed with Mg2+. We now report that the mitochondrial uncoupler carbonyl cyanide p-trifluoro-methoxyphenylhydrazone decreases ATP levels in rat pheochromacytoma (PC-12) cells differentiated with nerve growth factor and activates a neurofilament kinase, a tau kinase, and, unexpectedly, a tau phosphatase--either PP1 or PP2A. Such aberrant modulation of protein phosphorylation patterns could be the common biochemical basis for senile dementia and for Alzheimer disease and could explain the late-onset etiology of both conditions.

Targeted gene walking by low stringency polymerase chain reaction: assignment of a putative human brain sodium channel gene (SCN3A) to chromosome 2q24-31.

We have developed a low stringency polymerase chain reaction (LSPCR) to isolate the unknown neighboring region around a known DNA sequence, thus allowing efficient targeted gene walking. The method involves the polymerase chain reaction (PCR) with a single primer under conditions of low stringency for primer annealing (40 degrees C) for the first few cycles followed by more cycles at high stringency (55 degrees C). This enables the amplification of a targeted DNA fragment along with other nontargeted fragments. High stringency (55 degrees C) nested PCRs with end-labeled primers are then used to generate a ladder of radioactive bands, which accurately identifies the targeted fragment(s). We performed LSPCR on human placental DNA using a highly conserved sodium channel-specific primer for 5 cycles at 40 degrees C followed by 27 cycles at 55 degrees C for primer annealing. Subsequently, using higher stringency (55 degrees C) PCR with radiolabeled nested primers for 8 cycles, we have isolated a 0.66-kb fragment of a putative human sodium channel gene. Partial sequence (325 bp) of this fragment revealed a 270-bp region (exon) with homology to the rat brain sodium channel III alpha (RBIII) gene at the nucleotide (87%) and amino acid (92%) levels. Therefore, we putatively assign this sequence as a part of a gene coding the alpha-subunit of a human brain type III sodium channel (SCN3A). Using PCR on two human/rodent somatic cell hybrid panels with primers specific to this putative SCN3A gene, we have localized this gene to chromosome 2. Fluorescence in situ hybridization to human metaphase chromosomes was used to sublocalize the SCN3A gene to chromosome at 2q24-31. In conclusion, LSPCR is an efficient and sensitive method for targeted gene walking and is also useful for the isolation of homologous genes in related species.

Hyperphosphorylation of human TAU by brain kinase PK40erk beyond phosphorylation by cAMP-dependent PKA: relation to Alzheimer's disease.

Abnormal hyperphosphorylation of the cytoskeletal protein TAU is seen in the characteristic paired helical filaments [neurofibrillary tangles] of Alzheimer's disease [AD]. A recently described protein kinase, PK40erk, (1) a member of the ERK family of kinases, can produce in vitro many of the properties of Alzheimer-like hyperphosphorylated TAU. cAMP-dependent protein kinase A [PKA] phosphorylates TAU to a lesser extent; however, the product is not like the hyperphosphorylated TAU of AD in several important respects. We now report that in vitro PK40erk, a candidate for the enzyme responsible for TAU hyperphosphorylation in AD, will further phosphorylate TAU that was previously saturated by protein kinase A, provided that the concentrations of free uncomplexed ATP are low. Interestingly, the actions of different kinases on TAU are not independent, but may depend on the order in which they work on TAU; i.e., prior phosphorylation by PKA partially inhibits the action of PK40erk.

Localization of a putative human brain sodium channel gene (SCN1A) to chromosome band 2q24.

We have identified four putative human sodium channel gene sequences, 55 bp each, using the polymerase chain reaction (PCR) on total human placental DNA with primers specific for the cDNA sequence of the rat brain sodium channel I alpha (Scn1a) gene. One of these sequences was extended bidirectionally by genomic inverse-PCR to obtain a 1.6-kb fragment. Sequencing of this 1,556-bp fragment showed a 282-bp complete exon, which has 95% and 94% homology at the nucleotide and amino acid levels, respectively, with the rat Scn1a gene. We putatively assign this sequence as belonging to the gene coding the alpha-subunit of a human brain type I sodium channel (SCN1A). PCR on human x rodent somatic cell hybrids with primers derived from SCN1A localized this gene to chromosome 2. Fluorescence in situ hybridization to human metaphase chromosomes sublocalized the gene to chromosome band 2q24.

Brain protein kinase PK40erk converts TAU into a PHF-like form as found in Alzheimer's disease.

The novel protein kinase PK40 (1) was characterized by its ability to phosphorylate Lys-Ser-Pro sites in neurofilament and TAU proteins. PK40 is now recognized to be a member of the family of External-stimulus Regulated Kinases (ERKs) by its reactivity with ERK-specific antibodies and will therefore be called PK40erk. Bovine TAU or recombinant human TAU proteins can be hyperphosphorylated by PK40erk to produce the electrophoretic mobility shifts and certain immunochemical properties characteristic of PHF-TAU isolated from Alzheimer's disease brain tissue. PK40erk may play a crucial role in the etiology of this disease.

A mitochondrial DNA deletion in normally aging and in Alzheimer brain tissue.

By quantitative polymerase chain reaction (PCR) of total cellular DNA, the known 4977 bp deletion in human mitochondrial DNA (mtDNA delta 4977) was not detected in rapidly dividing tissue such as placenta and lymphocytes, nor in brain tissue from fetuses and in frontal cortex from two individuals 24 and 56 years old. However, in frontal cortex from individuals 71-95 years 0.13% deleted/undeleted mtDNA was found, with no significant difference between Alzheimer patients (0.14%) and age-matched controls (0.12%). We hypothesize that the age-related accumulation of this deletion (and other expected deletions) contributes to the down-regulation of adenosine triphosphate (ATP) production in neurons and other non-dividing cells, a fundamental mechanism common to aging and Alzheimer's disease.

Two novel kinases phosphorylate tau and the KSP site of heavy neurofilament subunits in high stoichiometric ratios.

We have identified, purified, and characterized two neurofilament/tau kinases from bovine brain, PK36 and PK40, with apparent Mr of 36,000 and 40,000 and with novel biochemical properties. A specially designed immunoassay for phosphorylated epitopes in neurofilament (NF) proteins was used in the early stages of the purification. Neither kinase is closely associated with the cytoskeleton. Both kinases phosphorylate bovine intermediate (NF-M) and heavy (NF-H) NF subunits and also bovine tau at the expected KSP sequences, though other sites cannot be ruled out. In human paired helical filaments, tau, phosphorylated at these same KSP sites, is a major characterized constituent. Neither kinase is activated by the usual second messengers. Tau and the above NF subunits are phosphorylated in high stoichiometric ratios. In the intermediate NF subunit, all the expected sites appear to be phosphorylated, but in the heavy NF subunit only 7 out of the greater than 40 expected sites can be phosphorylated by our kinases. We demonstrate that both kinases can induce considerable shifts of apparent Mr with SDS-PAGE for tau and, for the first time in vitro, also for the intermediate NF subunit. Interestingly, PK36 and particularly PK40 are strongly inhibited by an excess of free ATP. We propose that during normal aging, and in Alzheimer's disease, age-related mitochondrial dysfunction would reduce ATP levels, which in turn might release the neurofilament/tau kinase from inhibition with consequent paired helical filament formation.

Selective distribution of a novel tubulin in the developing and mature rat brain.

A monoclonal antibody, G8, was isolated which recognizes a form of tubulin (G8-tubulin) with a novel distribution in the rat brain. Immunoblots of rat brain homogenates and immunohistochemical staining of rat brain sections of various ages with G8 revealed a restricted and developmentally regulated distribution of the G8-tubulin. G8 staining was primarily found in granule cell dendrites in the dentate gyrus, in pyramidal cell apical dendrites in the hippocampus, and in Purkinje cell dendrites in the cerebellum. This pattern was much more selective than that observed with three other anti-tubulin antibodies. Relative abundance of G8-tubulin in the brain increases with age between postnatal day 1 (P1) and adulthood. The results suggest that G8 is specific for a novel tubulin form which shows a characteristic distribution in the rat brain. This distribution may indicate that the G8-tubulin possesses functional specificity.

A tubulin identified by a monoclonal antibody is not present in mitotic spindles.

A monoclonal antibody, G8, which recognizes a form of tubulin (G8-tubulin) with a novel distribution in Rat-1 cells and Potorous tridactylis kidney (Ptk-2) cells was isolated. G8 labeled the interphase cytoskeleton of Rat-1 fibroblasts but not mitotic spindles or midbodies. G8 also stained a fiber network in some but not all Ptk-2 interphase cells but did not label mitotic spindles or midbodies in these cells. G8-tubulin is the only identified tubulin known to be absent from these structures. This distribution may indicate that G8-tubulin possesses functional specificity.

Identification of a novel casein kinase activity in HeLa cell nuclei.

Three casein kinase activities have been resolved by column chromatography of HeLa cell nuclear extracts. In addition to casein kinases NI and NII, which have been described in other cell types, HeLa nuclei contain a third casein kinase activity which we have named NIII. NIII is a cyclic nucleotide-independent casein kinase which uses either Mg2+ or Mn2+ as a divalent cation, but is inhibited by increasing NaCl concentrations in the presence of Mg2+ and has optimal activity at 50 mM NaCl in the presence of Mn2+. In Mg2+, NIII uses only ATP as a phosphate donor, but in Mn2+ NIII transfers phosphate from either ATP or GTP. NIII phosphorylates the serine and threonine residues of casein, but does not phosphorylate phosvitin or calf thymus histones.

Age-related neurofilament phosphorylation in normal human brains.

An age-related phosphorylation of the 200kD neurofilament protein (NF200) has been observed in the axons of cerebellar basket cells of normal human fixed brain tissue from individuals older than 60 years, but not in younger individuals. The probe for this study was the monoclonal antibody SMI-34 which detects a phosphorylated epitope on NF200 which appears to be different from the more frequent NF200 phosphorylated epitope found by another monoclonal antibody, SMI-31. The SMI-31 epitope was detected in our specimens at all ages examined. In sections of brainstems from normal individuals older than 52 years, from Alzheimer's disease individuals older than 65 years and from older Down's syndrome individuals (greater than 56 years) certain axons in the medial longitudinal fasciculus and in the mesencephalic trigeminal tract react positively with SMI-34, the antibody which detects the "age-related" phosphorylation epitope. All our Alzheimer's disease specimens and all our Down's syndrome specimens, even the younger ones, show staining of these fibers with SMI-31. The biochemistry of the "age-related" phosphorylation of NF200 remains to be explained.

Diversity among Purkinje cells in the monkey cerebellum.

A monoclonal antibody (B1) produced against rat embryonic forebrain membranes shows specific and striking immunohistochemical staining of Purkinje cells in the monkey cerebellum in a pattern of broad parasagittal alternating bands of cells either possessing or lacking the B1 antigen. In addition, the neurons of the deep cerebellar nuclei and some neurons of the motor cortex and of the spinal cord also contain the B1 antigen. Neurons with the B1 antigen were also seen in the somatosensory cortex, the vestibular and cochlear nuclei, and the retina.