The effect of insulin deficiency on tau and neurofilament in the insulin knockout mouse

BBRC Biochemical and Biophysical Research Communications 334 (2005) 979–986 www.elsevier.com/locate/ybbrc

The effect of insulin deficiency on tau and neurofilament in the insulin knockout mouse Ruben Schechter a,b,*, Delia Beju a,b, Kenneth E. Miller b a

William K. Warren Medical Research Institute, University of Oklahoma Medical Health Science Center, Tulsa, OK 74107, USA b Department of Anatomy and Cell Biology, Oklahoma State University Center for Health Science, Tulsa, OK 74107, USA Received 9 June 2005 Available online 13 July 2005

Abstract Complications of diabetes mellitus within the nervous system are peripheral and central neuropathy. In peripheral neuropathy, defects in neurofilament and microtubules have been demonstrated. In this study, we examined the effects of insulin deficiency within the brain in insulin knockout mice mice (I( / )). The I( / ) exhibited hyperphosphorylation of tau, at threonine 231, and neurofilament. In addition, we showed hyperphosphorylation of c-Jun N-terminal kinase (JNK) and glycogen synthase kinase 3 b (GSK-3 b) at serine 9. Extracellular signal-regulated kinase 1 (ERK 1) showed decrease in phosphorylation, whereas ERK 2 showed no changes. Ultrastructural examination demonstrated swollen mitochondria, endoplasmic reticulum, and Golgi apparatus, and dispersion of the nuclear chromatin. Microtubules showed decrease in the number of intermicrotubule bridges and neurofilament presented as bunches. Thus, lack of insulin brain stimulation induces JNK hyperphosphorylation followed by hyperphosphorylation of tau and neurofilament, and ultrastructural cellular damage, that over time may induce decrease in cognition and learning disabilities.
2005 Elsevier Inc. All rights reserved. Keywords: Insulin; Tau; Neurofilament; JNK; ERK; GSK-3; Brain; Electron microscopy; Insulin knockout mice; Diabetes

In insulin type 1 (insulin dependent) and type 2 (noninsulin dependent) diabetes mellitus, one of the major complications is nervous system neuropathy [1]. More effort has been dedicated to study the etiology and pathology of peripheral neuropathy, but central nervous system neuropathy has also been described [2]. In peripheral neuropathy, alterations occur in neurofilaments with a concomitant decrease in nerve diameter and axonal shrinkage [3]. The data on central neuropathy are primarily descriptive and little has been investigated on its cause. The central nervous system neuropathy is characterized by decreased conduction velocity with changes in the EEG, cerebral atrophy (global subcortical and cortical atrophy), decrease in

*

Corresponding author. Fax: +1 918 561 8276. E-mail address: [email protected] (R. Schechter).

0006-291X/$ - see front matter
2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.07.001

cognition, and increased risk for stroke [2]. Furthermore, diabetes mellitus also has been associated with increased risk for AlzheimerÕs disease and other types of dementia [4]. In addition, Sharma et al. have described cerebral atrophy in young patients with type 1 diabetes mellitus who are otherwise healthy [5,2]. Animal studies may provide important clues into the cause of cerebral atrophy. For example, Shubert et al. [6] described the hyperphosphorylation of tau at threonine 231 in the brain of neuronal insulin receptor knockout mouse. This effect of insulin resistance was due to the inability of the neurons to activate Akt and inhibition of GSK-3 b at serine 9 [6]. Brain insulin sources can be from in situ production [7–9] or of pancreatic origin by crossing the blood–brain barrier [10]. Insulin synthesis has been demonstrated in the brain of fetal, newborn, and adult rats and mice by the presence of insulin mRNA and/or protein [7–9].

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Brain endogenous insulin [I(n)] promotes axon growth and neuronal differentiation [11,12]. I(n) promotes axonal growth by regulating neurofilament transport to the axon via ERKs [13]. In diabetes mellitus, neurofilaments are hyperphosphorylated promoting diabetic neuropathy [14]. Hyperphosphorylation of neurofilament induces a decrease in protein transport and promotes excessive accumulation of neurofilament within the axons [3]. In addition, insulin is related to the regulation of tau phosphorylation in cell cultures at serine 202 and threonine at 205, 231, and 180 [15]. Lack of insulin inhibition of GSK-3 b at serine 9 causes hyperphosphorylation of tau promoting microtubule depolymerization and possible induction of neurodegenerative diseases such as AlzheimerÕs [15]. The cytoskeleton proteins that form the microtubules and neurofilaments are present in a phosphorylated status during brain development to facilitate transport from the neuronal body to the axons and dendrites [16,17]. Mitogen activated protein kinases are a family of kinases that regulate different metabolic pathways within cells. ERK 1, ERK 2, and JNK are part of this family [3], and are known to phosphorylate tau and neurofilaments at different sites [18,3]. ERK 1 and 2 are activated by insulin [13,19] and JNK is regulated by insulin [20,21]. JNK can be phosphorylated by different stimuli: withdrawal of growth factors, environmental stress, and osmotic stress [20]. Ho et al. [22] showed that hyperglycemia in endothelial cells can induce the activation of JNK. JNK participates in different cellular processes, such as cell proliferation [23], and regulation of microtubule assembly and MAP activity [23]. A major complication of diabetes mellitus is central and peripheral neuropathy. Furthermore, diabetes mellitus is associated with the increasing risk for AlzheimerÕs disease or other forms of dementia, brain atrophy, and decreased cognition [2], but only a small number of studies show alterations in the cytoskeleton within neurons in the brain of diabetic patients. In the present study, we have investigated the phosphorylation status of tau and medium molecular weight neurofilament (NF-M) and ultrastructural alterations within the brain of insulin knockout mice [I( / )]. In addition, we studied the effects of lack of insulin in the phosphorylation of GSK-3 b at serine 9 and tyrosine at GSK-3 a and b, JNK and ERK. The I( / ) has been described previously by Duvillie et al. [24] and these mice develop diabetes mellitus soon after feeding with a survival of 3– 4 days.

Materials and methods Reagents. Insulin knockout mice were obtained from INSERM, Paris, France (generously provided by Drs. Jami and Deltour). Insulin 1 gene and insulin gene 2 mice were bred to obtain the insulin

knockout mice. Analysis of the pathology was performed previously [24]. The I( / ) developed diabetes mellitus after the first meal and were diagnosed by the presence of glycosuria (kit from Sigma, St. Louis, MO). Antibodies against phosphorylated tau were: mouse monoclonal AT-8 (serine 202, threonine 205), AT-180 (threonine 231), and AT-270 (threonine 181) from Innogenetics (Gent, Belgium), 12E8 (serine 262,356) a gift from Elan Pharm (Dublin, Ireland), and rabbit anti-Tau-1 that recognizes non-phosphorylated tau at epitopes 189– 207 from Sigma (St. Louis, MO). Other antisera used were: rabbit antiGSK-3 b serine 9 (New England Biolabs, Beverly, MA), mouse monoclonal anti-phosphotyrosine GSK a (tyrosine 279) and b (tyrosine 216) (UBI, Lake Placid, NY), rabbit anti-JNK to threonine 183 and tyrosine 185 (New England Biolabs, Beverly, MA), and rabbit anti-active MAPK that recognizes ERK 1 and 2 (Promega, Madison, WI). Mouse monoclonal anti-medium molecular weight neurofilament RMO-281 that recognizes phosphorylated neurofilament was from Zymed (San Francisco, CA) and guinea pig anti-porcine insulin antibody was from Linco (St. Louis, MO). Phosphatase inhibitor cocktail I and II, and protease inhibitor were from Sigma (St. Louis, MO), nitrocellulose paper was from S&S (Keene, NH), and PhastSystem and gels were from Pharmacia (Uppsala, Sweden). Western blots. Five brains were obtained from 36-h-old insulin knockout mice [I( / )] and wild type mice [I(+/+)]. The I( / ) mice developed diabetes mellitus after the first meal and were diagnosed by the presence of glycosuria (glucose >500 mg/dl). Brains were homogenated, as described by Lesor et al. [25] with some modifications, in a lysis buffer consisting of 10 mM Tris, pH 7.4, 2 mM EDTA, 50 ll Triton X-100 with phosphatase inhibitor cocktail I and II, and protease inhibitor. The homogenated brains were centrifuged at 70,000 RPM for 20 min and the protein concentration of the supernatant was measured using a bicinchoninic acid kit (BCA Protein Assay Kit, Pierce, Rockford, IL). For normalization of the Western blots, equal amount of total protein was used. Medium molecular weight neurofilament (NF-M) studies were performed using 7.5% gels, and tau, ERK 1 and 2, GSK, and JNK in 12.5% gels to study phosphorylation. Antibodies to tau phosphorylated epitopes AT-8 (serine 202, threonine 205), AT-180 (threonine 231), AT-270 (threonine 181) (Innogenetics), 12E8 (serine 262,356, gift Elan Pharm), and Tau-1 that recognize non-phosphorylated tau were used. Antibody to NF-M RMO-281 (Zymed) recognizes phosphorylated-dependent tail domain of the C-terminal of the NF-M. Antibodies were used to GSK-3 b serine 9 and tyrosine in GSK a and b, JNK to threonine 183 and tyrosine 185, and ERK antibody that recognizes both ERK 1 and 2. To normalize the Western blots, total protein was used at a final concentration of 200 lg/ml per band. Proteins were resolved in 10 mM Tris base, pH 8.0, 1 mM EDTA, 2.5% SDS, 5% b-mercaptoethanol, and 5% bromophenol blue and heated at 100 C for 5 min as described previously [13]. After separation, the proteins were transferred to a nitrocellulose membrane using the PhastSystem in a buffer of 25 mM Tris, pH 8.0, 192 mM glycine, and 20% methanol at 25 MA for 20 min. Samples from the I( / ) and wild type mice were studied employing the Protoblot II AP System (Promega). Molecular weight standards were from Novagen. In brief, after the transfer was completed, the membranes were dried at 37 C for 10 min and then washed in 20 mM Tris–HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween 20 (TBST). The membranes were treated with TBST plus 1% bovine serum albumin. Afterwards, the membranes were incubated with the corresponding primary antibody overnight at 4 C. The following day, the membranes were washed in TBST followed by incubation in the corresponding goat anti-mouse or rabbit second antibody conjugated with alkaline phosphatase for 30 min at room temperature. They were then washed in TBST two times for 5 min followed by wash in 20 mM Tris– HCl, pH 7.5, and 150 mM NaCl two times for 5 min. The membranes were incubated in Western blue stabilized for alkaline phosphatase as a chromogen. Quantification was performed by digitizing the bands on the membranes and Image Tool (UTHSCSA) was used for analysis of the bands.

R. Schechter et al. / Biochemical and Biophysical Research Communications 334 (2005) 979–986

Results The I( / ) mice became hyperglycemic soon after the first meal. Urine glucose was above 500 mg/dl whereas the normal animalsÕ urine glucose was <10 mg/dl. The mice produced 1 of 16 as a knockout and the I( / ) animals die between 3 and 5 days of life.

TAU

A Kd

Kd

Kd

Kd

12E8

AT8 46

50 35

AT270 50

AT180

46

50 35

Tau-1 50 35

46 s

B Kd

46

46 s

I+/+ I-/-

50 35

I+/+ I-/-

Electron microscopy. Samples of I( / ) and wild type mice hippocampus, olfactory bulb, and pancreas were processed for electron microscopy examination. Both hippocampus and olfactory bulb contain high concentration of insulin [7]. Hippocampi, olfactory bulb, and pancreas were dissected and fixed in cacodylate buffered 2% glutaraldehyde, followed by 1% aqueous osmium tetroxide, and embedded in PolyBed 812. Thin sections were incubated with guinea pig anti-insulin antibody (1/1000) and the immunoreactive sites for I(n) were visualized with protein A–colloidal gold complex (15 nm) as previously described [26]. In addition, other sections were analyzed for microtubule number, diameter, distance between and number of intermicrotubular bridges, and quantitated in a defined surface of 120 cm2. Distance between the microtubules and the diameter of microtubules were expressed in arbitrary units. Statistical analysis. Equal amount of total protein was used for normalizing the Western blot. Western blot bands were digitized and the densities were analyzed with Image Tool (UTHSCSA). The mean density and standard error of the mean (SEM) for I / and wild type mice are reported. Comparison of the means from I / and wild type animals was performed with the StudentÕs test and p < 0.05 was considered significant. The percent changes of I / from wild type also are reported.

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NEUROFILAMENT 225 150

Kd 170

s

I+ /+

I-/-

Fig. 1. This figure represents the Western blots studies of tau and neurofilament within the I( / ) and the wild type mouse. To normalize the Western blots, total protein was used at a final concentration of 200 lg/ml per band. (A) These Western blots represent the studies using different antibodies to tau phosphorylated sites: 12E8, AT-8, AT-270, AT-180, Tau-1. Note that only antibody AT-180 that recognizes threonine at 231 showed a significant increase in phosphorylation in the I( / ); p < 0.05. All antibodies recognized a unique band at 46,000 molecular weight. (B) This Western blot represents the study of the medium chain molecular weight neurofilament, note the increase in phosphorylation within the I( / ); p < 0.05. Statistical analysis was performed using the StudentÕs t test.

Western blot analysis Tau, as expected for the age of nervous system development, showed that all the target epitopes in the study were phosphorylated in the wild type animal brain. In the I( / ) brain, some of these epitopes, but not all, were hyperphosphorylated. Increased immunoreaction (49.66%) was only detected by phospho-dependent antibody AT-180 that reacts with threonine 231 (I( / ), 55.75 ± 4.50; wild type, 37.25 ± 5.61, p < 0.05) of tau according to the sequence of the large tau molecule (Fig. 1). Phospho-dependent antibodies AT-8 (I( / ): 39.80 ± 2.41; wild type: 41 ± 3.30), AT-270 (I( / ): 101.80 ± 6.47; wild type: 113.80 ± 7.99), 12E8 (I( / ): 182 ± 22.31; wild type: 144 ± 22.08) demonstrated immunoreaction in both wild type and I( / ), but no significant differences were observed (p > 0.05) (Fig. 1). Tau-1 antibody did not show a significant difference (p > 0.05; I( / ): 129 ± 3.97; wild type: 126 ± 1.24) (Fig. 1). The antibodies used for phosphorylated and unphosphorylated tau recognized a band of 46,000 molecular weight, as reported previously for the age of the animals used [16]. Neurofilament phosphorylation also was studied in the I( / ) mice to investigate the effect of the lack of insulin in the brain. Because of the age of the animal, we studied the phosphorylated status of the NF-M using

a phosphorylated-dependent antibody. The NF-M was significantly hyperphosphorylated in the I( / ) and, as expected, the wild type mouse brain showed phosphorylated NF-M (I( / ) 162 ± 10.66; wild type: 122.3 ± 8.53; p < 0.05), with an increase of 30.25% (Fig. 1). The phosphorylation status of ERK, JNK, and GSK a and b, known to be involved in the phosphorylation of tau and neurofilament, was investigated for the effect of deficiency of insulin (Fig. 2). JNK was phosphorylated in both wild type and I( / ) in the two isoforms (JNK 1 and 2), but a significant hyperphosphorylation was observed in the I( / ). JNK 1 (46,000 molecular weight) was found to be highly phosphorylated compared to the wild type mouse in the (I( / ): 200 ± 17.95; wild type: 128.3 ± 10.17; p < 0.05) with an increase of 55.88 %, whereas JNK 2 (56,000 molecular weight) was increased by 7.4 % (I( / ): 116 ± 7.83; wild type 108 ± 3.51; p < 0.05). Within the I( / ), JNK 1 was significantly hyperphosphorylated when compared to JNK 2 (JNK 1 I( / ): 200 ± 17.95; JNK 2 I( / ): 116 ± 7.83; p < 0.05). ERK 2 in the I( / ) did not show significant changes (p > 0.05) when compared to phosphorylation levels in the wild type (I( / ): 54.12 ± 6.19; wild type: 75 ± 7.49), but had tendency toward decreased phosphorylation. ERK 1 in the

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A

JNK

53 46

50 35

s C

GSK-3

I+/+

B

s D

(serine 9) 46

35

45 42

35

I-/-

50

ERK

50

I+/+

GSK-3

I-/(Tyr) 51 46

50 35

s

I+/+

I-/-

s

I+/+

I-/-

Fig. 2. This figure represents the Western blots studies of different kinases within the I( / ) and the wild type mouse. To normalize the Western blots, total protein was used at a final concentration of 200 lg/ml per band. (A) This Western blot represents the study of JNK, note the increase in phosphorylation within the I( / ); p < 0.05. Both isoforms were significantly hyperphosphorylated in I( / ), but the 46,000 molecular weight isoform was highly phosphorylated when compared to the 56,000 molecular weight within the I( / ) (p < 0.05). (B) This Western blot represents the study of ERK, note the decrease in phosphorylation within the I( / ), but only ERK 1 (45,000 molecular weight) showed a significant decrease in phosphorylation within the I( / ) when compared to the wild type (p < 0.05). Within the I( / ), ERK 1 phosphorylation was significantly decreased when compared to ERK 2 (p < 0.05). (C) This Western blot represents the phosphorylation studies of GSK-3 b serine 9, an increase of the serine 9 site was observed within the I( / ); p < 0.05. (D) This Western blot represents the phosphorylation studies of GSK-3 a and b tyrosine phosphorylation, no difference was found between I( / ) and wild type mouse; p > 0.05. Statistical analysis was performed using the StudentÕs t test.

I( / ) showed a significant decrease in phosphorylation when compared to the wild type reflecting a decrease of 28% (I( / ): 34 ± 3.39; wild type: 47.20 ± 4.18; p < 0.05). Within the I / ERK 1 phosphorylation was significantly decreased when compared to ERK 2 (p < 0.05). GSK-3 a and b tyrosine phosphorylation showed no significant changes between I( / ) and the wild type mouse (GSK-3 a I( / ): 148 ± 6.30; wild type: 148.4 ± 8.35; GSK-3 b I( / ): 90.20 ± 10.02; wild type: 98.80 ± 6.19, p > 0.05). Studies of GSK-3 b serine 9, that inhibits the effects of GSK-3 b on the phosphorylation of tau, showed an increase of the phosphorylation of this site of 29.8% (I( / ): 51.40 ± 2.46; wild type: 39.6 ± 1.20; p < 0.05). Electron microscopy We studied the ultrastructural effects on microtubules and neurofilament in the I( / ) mice compared to the wild type mice. The number of bridges between the microtubules, microtubule diameter, total number of microtubules, the distance between microtubules, and the distribution of neurofilaments were quantitated in a defined area of 120 cm2. All measurements represent arbitrary units. Both the hippocampus and the olfactory bulb demonstrated similar results. The electron microscope microphotograph of the hippocampus serves as an example.

The ultrastructure (Fig. 3) of the microtubules showed a significant decrease (p < 0.05) in the number of the bridges between these structures (wild type: 17.25 ± 1.64; I( / ): 9.00 ± 1.108), but the distance within the microtubules was maintained (p > 0.05) (wild type: 50.0 ± 4.54; I( / ): 38.08 ± 5.19). The number of microtubules also was similar between the I( / ) and wild type mice (p > 0.05) (wild type: 7.75 ± 0.49; I( / ): 7.91 ± 0.48). The diameter of the microtubules in the I( / ) mice was significantly decreased compared to the wild type mice (wild type: 37.94 ± 0.94; I( / ): 31.16 ± 0.43; p < 0.05). The microtubules in the I( / ) mice had an aberrant distribution and appeared to be fractured (Fig. 3). Additionally, a web-like formation occurred sporadically within the neurons (Fig. 3). Neurofilaments were distributed in bunches within neuronal prolongations, i.e., axon hillocks and initial segments of dendrites (Fig. 3). Ultrastructural studies also revealed damage to organelles, the endoplasmic reticulum, Golgi apparatus, and swollen mitochondria with loss of the cristae (Fig. 3). The neuronal bodies also were swollen and the chromatin was dispersed with a loss of density (Fig. 3). Positive insulin immunoreaction within the rough endoplasmic reticulum of the wild type mice hippocampal neurons was detected (Fig. 3). The wild type mice pancreas also showed positive immunoreaction within the granules of b cells (Fig. 3).

Discussion Diabetes mellitus type I in young children is associated with decreased cognitive abilities and brain atrophy [5]. Type I and type II diabetes mellitus show complications over time that include central and peripheral neuropathy, nephropathy, and stroke [1,2], and an association with AlzheimerÕs disease [4]. The studies of central diabetic neuropathy are more of clinical observations, whereas in peripheral neuropathy it has been shown that neurofilaments are hyperphosphorylated by JNK and ERKs inducing a loss of axon caliber and axonal retraction [3]. Studies investigating the status of microtubules in diabetes are minimal [15,25]. Tau and neurofilament play a role in neuron growth and differentiation during brain development [27]. In the current report, chronic studies were not possible to be performed because the I( / ) mice die within 3–4 days of life and an attempt to maintain the animals in an insulin protocol fails. GSK-3 b, JNK and ERK phosphorylate neurofilaments, and tau [27,25,18,3,5]. Our data demonstrated that tau was hyperphosphorylated at threonine 231 in I( / ), whereas the phosphorylated epitopes demonstrated by AT-8 (serine 202, threonine 205), AT-270 (threonine 181), 12E8 (serine 262, 356), and total tau

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Fig. 3. This figure represents the ultrastructural studies within the I( / ) and the wild type mouse within the hippocampus. (A) This microphotograph represents the immunostain of insulin within the wild type mouse pancreas, note the positive immunoreaction within the b cells, 44,000·. (B) This microphotograph represents the immunostain of insulin within the I( / ) pancreas, note the absences of immunoreaction within the b cells, 44,000·. (C) Immunolocalization of neuronal insulin (arrows) within the rough endoplasmic reticulum (ER) of a neuron, 45,000·. (D) A neuron within the wild type mouse. Note the normal organelles and normal chromatin distribution. N, nucleus; arrows, mitochondria, 12,800·. (E) This microphotograph represents the I( / ) hippocampus neuron. Note. (1) Abnormally dispersed chromatin; N, nucleus; (2) swollen cytoplasm and organelles. The mitochondria (arrow) show loss of the cristae, 12,600·. (F) This microphotograph represents the wild type mouse neuron. Note the normal distribution of the microtubules and number of bridges (arrows), 290,000·. (G) This microphotograph represents the I( / ) neuron. Note the aberrant distribution and fractured (arrow) microtubules. Note the decrease in the number of bridges between the microtubules (arrowheads) 290,000·. (H) This microphotograph represents the I( / ) neuron. Note the aberrant distribution of the neurofilaments (oval), 290,000·. (I) This microphotograph represents the I( / ) neuron. Note the web-like structure seen within the neurons (oval), 290,000·.

showed no significant difference when compared to the control wild type mouse. These phosphorylated amino acid sites correspond to the newborn tau status, in which tau is highly phosphorylated [28]. The reaction with antibody AT-8 that recognizes serine 202 and threonine

205 was weak, whereas antisera for the other epitopes showed strong reactions. The molecular weight of tau identified in the brain of the newborn mice wild type or I( / ) agrees with the molecular weight described previously by Takuma et al. [16] for newborn age. NF-

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M was found to be hyperphosphorylated in the I( / ), similar to the peripheral nervous system in which aberrant neurofilament phosphorylation is found during diabetic neuropathy [3]. The detection of only medium molecular weight neurofilament is consistent with the developmental age of the animal used in this study. The use of antibody to the high molecular weight (SMI-311) fails to detect neurofilament (data not shown). We studied the effects of the lack of insulin on the phosphorylation status of kinases known to have a role in the phosphorylation of tau and neurofilament [27,25,18,3,5] within the brain. In this study, we investigated GSK-3 b, ERK 1 and 2, and JNK 1 and 2. JNKs, and specifically JNK 1, were hyperphosphorylated when compared to the wild type mice. GSK-3 b was phosphorylated at serine 9, indicating that the kinase activity was inhibited. The activity of GSK-3 b is regulated by insulin by phosphorylation of serine 9 inhibiting the kinase activity [15]. ERK 1 and 2 have been demonstrated to be regulated by insulin [13,19] and ERKs phosphorylate tau and neurofilament [18,27,3]. Our study showed that ERK 1 phosphorylation was decreased and no changes occurred in ERK 2. These data demonstrate that insulin regulates ERK 1 within the brain, but another growth factor or factors may regulate ERK 2. Most recently, Schubert et al. [29] showed that tau was hyperphosphorylated in the insulin receptor substrate-2 knockout mouse. These insulin resistant mice presented increased phosphorylation on serine 202 and showed that GSK-3 b was phosphorylated at serine 9. Their study showed that GSK-3 b activity, in spite of insulin resistance, was inhibited. The authors, however, did not investigate other kinases that may be involved. Schubert et al. also studied the effects of insulin resistance in the brain by creating a brain/neuron-specific insulin receptor knockout (NIRKO [6]). In these animals, GSK-3 b activity, phosphorylation of serine 9, was not inhibited showing that tau was hyperphosphorylated at threonine 231. These two studies related to insulin resistance showed that there are differences within the inability of insulin to stimulate the insulin receptor or not. In our case, the data are most close to the NIRKO animals. This may be because other growth factors may be able to stimulate signal transduction substrates common to insulin stimulation. It is also interesting that in the I / mice, threonine 231 was phosphorylated in tau, a site suggested as a marker for AlzheimerÕs disease from spinal fluid [30]. This may imply that the phosphorylation of threonine 231 can be an early change for the detection of neurodegenerative disease associated with diabetes mellitus. Ultrastructural studies showing altered organelles and disrupted cytoskeleton revealed that the neurons undergo severe stress from the lack of insulin. Johnson et al. [31] reported in central nervous system the loss

of neurons in diabetes mellitus. Other studies demonstrate in diabetes mellitus animals similar results to our current work [32]. In the peripheral neuropathy, alterations in the morphology of the axon occur by decreasing the number of microtubules and neurofilaments and axon caliber [33]. Our ultrastructural observations are consistent with a high level of stress to the neurons as exemplified by swollen mitochondria, endoplasmic reticulum and Golgi apparatus, and a disorganized nucleus. These results are similar to Luo et al. [32] in which swollen mitochondria, endoplasmic reticulum, and Golgi apparatus were found in brains of rats treated with streptozotocin. The phosphorylation of the tail domain of NF-M allows for the stabilization of the filament by promoting lateral extension of the sidearm that increases neurofilament spacing, axon caliber, and conduction velocity of the nerve [34]. Hyperphosphorylation of neurofilament promotes aberrant accumulation and decrease in the slow transport of neurofilament [3]. In our current work, NF-M was detected by the RMO-281 antibody that recognizes the phosphorylated tail domain of the C-terminal of the neurofilament [33]. This aberrant neurofilament phosphorylation may decrease the transport and assembly of the neurofilament [3]. Scott et al. [33] showed that the number of neurofilaments in the axon of peripheral nerve is reduced in diabetes mellitus induced by streptozotocin. In the same study, a reduction in the number of microtubules occurred in the peripheral nervous system [33]. Fernyhough et al. [3] demonstrated in the peripheral nerve that JNK and ERK 1 and 2 were involved in the hyperphosphorylation of neurofilament. In our work, only JNK was hyperphosphorylated, particularly the 46,000 molecular weight, ERK phosphorylation was reduced, and GSK-3 b activity was inhibited. Furthermore, neurofilaments are hyperphosphorylated in brain and spinal fluid of patients with AlzheimerÕs disease and other neurodegenerative disorders [35,36]. The hyperphosphorylation of NF-M and tau in the I / suggests that diabetes mellitus is associated with neurodegenerative disorders as has been proposed [4,36]. The kinases studied in the current work, JNK, ERKs, and GSK-3 b, are regulated by insulin and, in turn, these three kinases regulate the phosphorylation of tau and neurofilament. JNK, ERK, and GSK-3 b phosphorylate neurofilament at different motifs of the tail domain [34] and tau is phosphorylated at different sites [18]. GSK-3 b, JNK, and ERK phosphorylate tau at epitopes studied in this study: serine 202, 205 and threonine 231, 181 in vitro [18]. Only JNK was found to be hyperphosphorylated in the I( / ). JNK is regulated by insulin [20,21] and can be phosphorylated by different stress stimuli, such as hyperglycemia and growth factor withdrawal [20]. JNK has a role in nor-

R. Schechter et al. / Biochemical and Biophysical Research Communications 334 (2005) 979–986

mal cell function and is required for microtubule maintenance [23]. GSK-3 b activity in normal cells is inhibited by insulin by phosphorylation of serine 9 and ERK is activated by insulin via insulin receptor substrate 1 activity (IRS-1) [19,15]. JNK has a direct effect on the insulin cascade by inhibiting IRS-1 activity [37] consequently blocking insulin regulation of ERK and GSK-3 b. In the present study, GSK-3 b was phosphorylated at serine 9 and ERK phosphorylation was decreased. This may imply, in the case of GSK-3 b, that other growth factors are involved in the regulation of these kinases. Also, ERK may lose activity when cells lack insulin stimulation with the addition that JNK inhibit IRS-1 activity [37]. Insulin like growth factor 1 (IGF-1) has been shown to activate a similar cascade as that of insulin [15]. We speculate that IGF-1 inhibits GSK-3 b in I( / ), but was not able to maintain normal glucose metabolism. The etiology of diabetes mellitus neuropathy is controversial. Lack of insulin, hypoglycemia, hyperglycemia, and temporal combinations of all three have been postulated to be the cause of the pathological alterations seen in diabetes neuropathy [38–41]. The I( / ) mice lack insulin and develop hyperglycemia. The hyperphosphorylation of JNK in the I( / ) can be attributed to the stress condition of hyperglycemia. Hyperglycemia, a consequence of insulin deficiency, induces JNK hyperphosphorylation which in time hyperphosphorylates tau and neurofilament. In contrast, ERK was not hyperphosphorylated, but had decreased activity as a direct effect of lack of insulin and GSK-3 b showed phosphorylation of the inhibitory site, serine 9. The mitochondria and other organelles were swollen may be as a consequence of the hyperglycemia and increased neural glucose intake by glucose transporter 3 that respond to glucose concentration [43]. We hypothesize that lack of insulin promotes chemical and ultrastructural changes in the I( / ) neuronal cytoskeleton inducing dysfunction that influences cellular transport promoting diabetic neuropathy, neuronal differentiation affecting learning, and cognitive memory, and, in older patients, the onset of AlzheimerÕs type dementia associated with diabetes mellitus. These data show that insulin has a role within the brain in maintaining a balance in phosphorylation of the neuronal cytoskeleton and that the lack of insulin disrupts this balance. We hypothesize that pancreatic insulin with its short half-life (1–2 min) is a difficult source to constantly maintain the cytoskeleton equilibrium, especially when pancreatic insulin is secreted by glucose stimulation. This does not explain how pancreatic insulin will be available constantly to neurons. Alternatively, local production of insulin has been demonstrated within the brain [7–9]. Thus, a de novo production of insulin by all or a subset of neurons and the choroid plexus [42] could provide a constant insulin source for the nervous system.

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