Enoxaparin ͉ neurodegeneration ͉ transgenic mouse ͉ mitochondria ͉ Lovenox H untington's disease (HD) has onset usually between 35 and 50 years with chorea and psychiatric disturbances and gradual but inexorable intellectual decline to death after 15-20 years (1). Neuropathological analysis reveals selective and progressive neuronal loss in the striatum (1), particularly affecting the GABAergic medium spiny neurons (MSN). At the molecular level, the cause of HD is a polyglutamine expansion (exp) in the amino terminus of huntingtin (Htt), a 350-kDa ubiquitously expressed cytoplasmic protein (2). Despite significant progress, cellular mechanisms that link the Htt exp mutation with the disease are poorly understood (3).A number of transgenic HD mouse models have been generated that reproduce many HD-like features (4). In the yeast artificial chromosome (YAC128) mouse model, the full-length human Htt protein with polyglutamine exp (128Q) is expressed under the control of its endogenous promoter and regulatory elements (5). The onset of a motor deficit before striatal neuronal loss in the YAC128 mouse model accurately recapitulates the progression of HD (5). Thus, the YAC128 mouse model is ideal for understanding the cellular mechanisms that lead to neurodegeneration in HD, as well as for validating potential therapeutic agents.Previous studies demonstrated that Htt exp facilitates activity of the NR2B subtype of NMDA receptors (NMDARs) (6-8) and the type 1 inositol 1,4,5-trisphosphate receptors (InsP 3 R1) (9). A connection between disturbed Ca 2ϩ signaling and neuronal apoptosis is well established (10, 11), and we therefore proposed that Htt exp -induced Ca 2ϩ overload results in degeneration of MSN in HD (12). To test this hypothesis, we analyzed Ca 2ϩ signals and apoptotic cell death in primary cultures of MSN from the YAC128 mice. Our results provide further support to the hypothesis that disturbed Ca 2ϩ underlies neuronal cell death in HD (12) and allowed us to identify a number of potential therapeutic targets for HD treatment. Materials and MethodsPrimary Neuronal Cultures. Generation and breeding of YAC18 and YAC128 transgenic mice (FVBN͞NJ background strain) are described in refs. 5 and 13. Heterozygous male YAC128 or YAC18 mice were crossed with the wild-type (WT) female mice and resulting litters were collected at postnatal days 1-2. The pups were genotyped by PCR with primers specific for exons 44 and 45 of human Htt gene and the medium spiny neuronal (MSN) or hippocampal neuronal (HN) cultures of WT, YAC18, and YAC128 mice were established and maintained as described in ref. 9.Ca 2؉ Imaging Experiments. Fura-2 Ca 2ϩ imaging experiments with 14-to 16-DIV (days in vitro) MSN cultures were performed as described in ref. 9, using a DeltaRAM illuminator, an IC-300 camera, and IMAGEMASTER PRO software (all from PTI, South Brunswick, NJ). The cells were maintained in artificial cerebrospinal fluid (aCSF) (140 mM NaCl͞5 mM KCl͞1 mM MgCl 2 ͞2 mM CaCl 2 ͞10 mM Hepes, pH 7.3) at 37°C during measurements (PH1 heat...

The inositol (1,4,5)-trisphosphate receptor (InsP3R) mediates Ca2+ release from intracellular stores in response to generation of second messenger InsP3. InsP3R was biochemically purified and cloned, and functional properties of native InsP3-gated Ca2+ channels were extensively studied. However, further studies of InsP3R are obstructed by the lack of a convenient functional assay of expressed InsP3R activity. To establish a functional assay of recombinant InsP3R activity, transient heterologous expression of neuronal rat InsP3R cDNA (InsP3R-I, SI− SII+ splice variant) in HEK-293 cells was combined with the planar lipid bilayer reconstitution experiments. Recombinant InsP3R retained specific InsP3 binding properties (K d = 60 nM InsP3) and were specifically recognized by anti–InsP3R-I rabbit polyclonal antibody. Density of expressed InsP3R-I was at least 20-fold above endogenous InsP3R background and only 2–3-fold lower than InsP3R density in rat cerebellar microsomes. When incorporated into planar lipid bilayers, the recombinant InsP3R formed a functional InsP3-gated Ca2+ channel with 80 pS conductance using 50 mM Ba2+ as a current carrier. Mean open time of recombinant InsP3-gated channels was 3.0 ms; closed dwell time distribution was double exponential and characterized by short (18 ms) and long (130 ms) time constants. Overall, gating and conductance properties of recombinant neuronal rat InsP3R-I were very similar to properties of native rat cerebellar InsP3R recorded in identical experimental conditions. Recombinant InsP3R also retained bell-shaped dependence on cytosolic Ca2+ concentration and allosteric modulation by ATP, similar to native cerebellar InsP3R. The following conclusions are drawn from these results. (a) Rat neuronal InsP3R-I cDNA encodes a protein that is either sufficient to produce InsP3-gated channel with functional properties identical to the properties of native rat cerebellar InsP3R, or it is able to form a functional InsP3-gated channel by forming a complex with proteins endogenously expressed in HEK-293 cells. (b) Successful functional expression of InsP3R in a heterologous expression system provides an opportunity for future detailed structure–function characterization of this vital protein.

The inositol 1,4,5-trisphosphate receptor (InsP 3 R) 1 plays a key role in intracellular Ca 2ϩ signaling (1). InsP 3 R have been biochemically purified, cloned, and functional properties of these channels were extensively studied (1-4). Planar lipid bilayer reconstitution technique proved useful for studies of functional properties and modulation of InsP 3 R (4). Modulation of cerebellar InsP 3 R by cytosolic Ca 2ϩ (5) and ATP (6) has been previously characterized by this method. Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) plays a major signaling role in cells (1). Hydrolysis of PIP 2 by phospholipase C (PLC) leads to production of inositol 1,4,5-trisphosphate (InsP 3 ) and Ca 2ϩ release from intracellular stores via activation of InsP 3 R. PIP 2 by itself can play a regulatory role in cells. Effects of PIP 2 on structural organization of cellular cytoskeleton are well established (7). More recently direct functional interaction between skeletal muscle ryanodine receptor (8), Na ϩ /Ca 2ϩ exchanger (9), and inward rectifier K ϩ channels (10) has been demonstrated. The emerging paradigm is that PIP 2 by itself may be a signaling messenger.We set out to test the hypothesis that PIP 2 may interact directly with the InsP 3 R. To investigate InsP 3 R-PIP 2 interaction, we reconstituted rat cerebellar InsP 3 R into planar lipid bilayers and tested response of these channels to addition of specific monoclonal anti-PIP 2 antibody (11), exogenous PIP 2 , and newly synthesized water-soluble PIP 2 analog dioctanoyl-(4,5)PIP 2 . Based on our data, we concluded that cerebellar InsP 3 R forms a stable inhibitory complex with PIP 2 that can be disrupted by anti-PIP 2 antibody. Membrane insertion of PIP 2 is not required for interaction with the InsP 3 R. We concluded that PIP 2 most likely interacts with InsP 3 binding site of the InsP 3 R. A novel model of compartmentalized Ca 2ϩ signaling is proposed based on results obtained in the study. EXPERIMENTAL PROCEDURESPlanar Lipid Bilayer Experiments-Rat (Sprague-Dawley; 4 -5 weeks old) cerebellar microsomes were isolated essentially as described previously for canine preparation (5, 6, 12) and stored frozen at Ϫ70°C. Planar lipid bilayers were formed from PE:PS (3:1) synthetic lipid mixture in decane on the small (100 -200 m in diameter) hole in Teflon film separating two chambers 3 ml each (cis and trans). Prior to formation of the bilayer the hole was prepainted with PC:PS mixture (3:1). Rat cerebellar InsP 3 R were incorporated into the bilayer by microsomal vesicle fusion as described previously for canine preparation (5, 6, 12). Single channel currents were recorded at 0 mV transmembrane potential using 50 mM Ba 2ϩ dissolved in HEPES (pH 7.35) in the trans (intraluminal) side as a charge carrier (12). In most of the experiments (standard recording conditions of InsP 3 R activity), the cis (cytosolic) chamber contained 110 mM Tris dissolved in HEPES (pH 7.35), 0.2 M free Ca 2ϩ (5) buffered with 1 mM EGTA and 0.7 mM CaCl 2 , and 1 mM Na 2 ATP (6). Unless indicated otherwise ...

Neuropathy with antibodies against myelin-associated glycoproteins (MAG/SGPG-N) and hereditary sensorimotor neuropathy type 1 (HMSN1) are characterized by chronic demyelination with little conduction block. Electrodiagnostic studies suggest that in HMSN1 conduction slowing occurs uniformly along the nerve, whereas in MAG/SGPG-N it is predominantly distal. Some but not all previous reports have shown that the terminal latency index (TLI) was useful to distinguish MAG/SGPG-N from chronic idiopathic demyelinating polyneuropathy. We compared median TLI from 21 patients with MAG/SGPG-N with those obtained from 26 patients with HMSN1, 20 with HMSN2, and 12 healthy volunteers. All patients with TLI <0.26 had MAG/SGPG-N, and all patients with TLI > or =0.32 had HMSN1. In the remaining patients with intermediate TLI values, ulnar distal motor latency (DML) aided in differentiation between MAG/SGPG-N and HMSN1 with an overall sensitivity of 100% and specificity of 98%. In conclusion, median TLI in combination with ulnar DML can further guide the demyelinating neuropathy evaluation toward hereditary or autoimmune causes.

We hypothesized that the corticospinal system undergoes functional changes in long-term polio survivors. Central motor conduction times (CMCTs) to the four limbs were measured in 24 polio survivors using transcranial magnetic stimulation (TMS). Resting motor thresholds and CMCTs were normal. In 17 subjects whose legs were affected by polio and 13 healthy controls, single- and paired-pulse TMS was used to assess motor cortex excitability while recording from tibialis anterior (TA) muscles at rest and following maximal contraction until fatigue. In polio survivors the slope of the recruitment curve was normal, but maximal motor evoked potentials (MEPs) were larger than in controls. MEPs were depressed after fatiguing exercise. Three patients with central fatigue by twitch interpolation had a trend toward slower recovery. There was no association with symptoms of post-polio syndrome. These changes occurring after polio may allow the motor cortex to activate a greater proportion of the motor neurons innervating affected muscles.

Evidence suggests that sensory loss may occur in a proportion of patients affected by poliomyelitis. We hypothesize that sensory problems may be a lasting sequela in some polio survivors. Sensory pathways in polio survivors were evaluated clinically and electrophysiologically using sensory evoked potentials (SEPs). Patients with sensory deficits or abnormal SEPs were further evaluated by magnetic resonance imaging (MRI). Twenty-two patients were studied. The mean age was 64.7 years (age range: 56-81 years). Clinically, sensory impairments were found in 4 patients. Upper limb SEPs were normal. Lower limb SEPs were abnormal in 10 patients. In 1 patient, clinical and electrographic findings correlated with a patch of atrophy in the spinal cord, as shown by MRI. Sensory derangements may be found in a proportion of aging polio survivors. SEP studies may add sensitivity when evaluating sensory function in this cohort. It remains unclear whether these sensory abnormalities are related to remote poliomyelitis. Further studies are necessary.

Patients with B-cell chronic lymphocytic leukemia (CLL) have an increased risk of second malignancy and may develop diffuse large-cell non-Hodgkin's lymphoma (DLCL) also known as Richter's syndrome (RS). Only seven cases of isolated brain RS without evidence of systemic lymphoma have been reported to date. We describe a case of isolated DLCL of the brain in a 58-year-old female patient with a 3 year history of B-cell CLL. The patient presented with falls due to left leg paresis and showed non-specific neuroimaging findings. Brain biopsy confirmed the diagnosis of DLCL and CLL restaging failed to demonstrate evidence of RS outside the CNS. The patient was treated with whole brain radiation therapy and was discharged 4 weeks later on weekly rituximab infusions. This report extends the previous experience by providing a detailed clinical, neuroradiological and pathological description of isolated RS of the brain. Early identification and timely CNS directed treatment may alter morbidity and positively influence quality of life.

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