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Journal of Lipid Research, Vol. 48, 1857-1872, August 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

The pathologies associated with functional titration of phosphatidylinositol transfer protein activity in mice

James G. Alb, Jr.^*, Scott E. Phillips^*, Lindsey R. Wilfley, Benjamin D. Philpot^,,** and Vytas A. Bankaitis^1,*

^* Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7090
Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7090
Neuroscience Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7090
^** Neurobiology Curriculum, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7090

Published, JLR Papers in Press, May 24, 2007.

¹ To whom correspondence should be addressed. e-mail: vytas{at}med.unc.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Phosphatidylinositol transfer proteins (PITPs) bind phosphatidylinositol (PtdIns) and phosphatidylcholine and play diverse roles in coordinating lipid metabolism/signaling with intracellular functions. The underlying mechanisms remain unclear. Genetic ablation of PITP in mice results in neonatal lethality characterized by intestinal and hepatic steatosis, spinocerebellar neurodegeneration, and glucose homeostatic defects. We report that mice expressing a PITP selectively ablated for PtdIns binding activity (Pitp^T59D), as the sole source of PITP, exhibit phenotypes that recapitulate those of authentic PITP nullizygotes. Analyses of mice with graded reductions in PITP activity reveal proportionately graded reductions in lifespan, demonstrate that intestinal steatosis and hypoglycemia are apparent only when PITP protein levels are strongly reduced (90%), and correlate steatotic and glucose homeostatic defects with cerebellar inflammatory disease. Finally, reconstitution of PITP expression in the small intestine substantially corrects the chylomicron retention disease and cerebellar inflammation of Pitp^0/0 neonates, but does not rescue neonatal lethality in these animals. These data demonstrate that PtdIns binding is an essential functional property of PITP in vivo, and suggest a causal linkage between defects in lipid transport and glucose homeostasis and cerebellar inflammatory disease. Finally, the data also demonstrate intrinsic neuronal deficits in PITP-deficient mice that are independent of intestinal lipid transport defects and hypoglycemia.

Supplementary key words signaling • phospholipids • lipoproteins • neurodegeneration

Abbreviations: CRD, chylomicron retention disease; ER, endoplasmic reticulum; EtOH, ethanol; fEPSP, field excitatory postsynaptic potential; GFAP, glial fibrillary acidic protein; MAMA, mismatch amplification mutation assay; PAC, P1 artificial chromosome; PITP, phosphatidylinositol transfer protein; PtdCho, phosphatidylcholine; PtdIns, phosphatidylinositol; TG, triglyceride

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The physical compartmentation of signal transduction to specific microdomains of membrane surfaces is a common property of all eukaryotic cells, and various mechanisms are applied toward achieving this end (1–5). One such mechanism involves the spatially and temporally regulated metabolic channeling of phosphatidylinositol (PtdIns) and/or phosphatidylcholine (PtdCho) monomers by phosphatidylinositol transfer proteins (PITPs). This PITP-mediated channeling is itself linked to specific physiological outcomes (6–8). Prime among these are the roles of PITPs in coordinating various interfaces between lipid metabolism and membrane trafficking reactions. PITPs fall into two unrelated structural classes: the Sec14-like PITPs and the so-called class 1 metazoan PITPs (9–12). The Sec14-like PITPs are better understood because detailed functional analyses have been more accessible for these proteins. The metazoan PITPs are poorly characterized from a functional point of view.

Mammals express four known class 1 PITP forms: PITP, PITPß, an alternative spliceoform of PITPß, and rdgBß (13–18). Data that address in vivo functions for PITP and PITPß are building. These proteins are encoded by distinct genes but are highly similar, sharing 77% and 95% primary sequence identity and similarity, respectively. The extensive homology notwithstanding, available data suggest that PITP and PITPß execute distinct roles in mammals. First, these proteins exhibit distinct intracellular distributions. PITP represents a cytosolic/nuclear protein, whereas PITPß is localized predominantly to the trans-Golgi network of mammalian cells [as is its alternative spliceoform (17)]. Second, PITPß binds the ceramide-based phospholipid sphingomyelin, whereas PITP does not (15, 17, 19). Finally, the consequences of the genetic ablation of each protein are different (20, 21). The functional nonredundancy of PITP and PITPß conforms to the general theme that PITPs are tightly coupled to specific physiological functions in cells (6–8). How PITPs couple individual PtdIns and PtdCho binding activities to biological functions remains to be determined.

Genetic ablation of the PITP structural gene in mice has interesting consequences. Pitp^0/0 progeny develop to term, but expire within several days after birth from a complex disease course that includes defects in dietary fat and -tocopherol transport from the small intestine [chylomicron retention disease (CRD)], liver steatosis, hypoglycemia, inflammation of cerebellum and hindbrain, and spinocerebellar neurodegenerative disease (21). These pathologies raise a number of questions. First, which biochemical properties of PITP are relevant for in vivo function (i.e., are the PtdIns and PtdCho binding activities differentially used in vivo, or are these themselves coupled?). Second, which of these pathologies are causally linked, and which derive from independent insults? It is suggested the intestinal defects and hypoglycemia are linked, and that these defects provoke the cerebellar and hindbrain neurodegenerative diseases (21). Resolution of these questions is key to an understanding of PITP function in mammalian systems.

Here, we show that PtdIns binding is an essential functional property of PITP in mammals. It is further demonstrated that intestinal steatosis and hypoglycemia require manifest reductions in PITP activity, and that these pathologies correlate with cerebellar inflammatory disease. Finally, the data indicate that reconstitution of PITP expression in the small intestine alleviates the CRD and cerebellar inflammatory disease of Pitp^0/0 neonates, but fails to effectively rescue the neonatal lethality of these animals. This failure is accompanied by exacerbations in microvesicular steatosis of the liver, a result consistent with the idea that PITP regulates the trafficking of luminal lipid cargos.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Mouse genotyping
Wild-type and Pitpa::NEO alleles were diagnosed from tail clip genomic DNA templates using a three-primer PCR assay described previously (21). To screen for the vibrator allele (Pitp^vb), a hypomorphic mutation that reduces the expression of wild-type PITP by 80%, an intracisternal A particle retrotransposon three-primer PCR assay was used (22). The allele-specific forward primers for the wild-type and vibrator alleles were Ham-For (5'-AGTGATCGGGACTTTTGTTTCC-3') and intracisternal A particle retrotransposon-1 (5'-AACGCGTCTAATAACACTTGTG-3'), respectively. A common reverse primer was used, Ham-Rev (5'-CCAAAAGGACTGCCCAGTCATTTTTCCCC-3'). The vibrator primer sequences were provided courtesy of Bruce Hamilton (University of California at San Diego).

To generate a PITP transgene whose expression is limited to the duodenum, plasmid pNSE-EX4 (23) (a gift of Greg Cox, Jackson Laboratories, Bar Harbor, ME) was cleaved with XhoI to remove the existing neuronal enolase promoter, and the intestinal fatty acid binding promoter (P_IFABP) (24) was introduced in its stead. The PITP open reading frame cDNA was generated by PCR as a 5'-HindIII-EcoRI-3' fragment and correspondingly subcloned into the P_IFABP-pNSE-EX4 vector to yield plasmid pFabpiPITP. The resultant plasmid was linearized by digestion with restriction endonuclease SalI and injected into C57BL/6JxC3H hybrid pronuclei at the University of North Carolina Animals Model Core to generate transgenic murine founder lines. Transgenic founders were identified with a PITP cDNA-specific set of primers: Alpha-For (5'-GCAAAGTTCCCACGTTTGTTCG-3') and Alpha-Rev (5'-CTCTTGCTTATGTATAAAGTTTTCC-3'). These primers support the synthesis of a diagnostic 450 bp PCR product. Three independent founders were crossed to C57BL/6J PITP^+/0 mice to generate Pitp^+/0;Tg(P_IFABP::PITP) lines.

Generation of a PtdIns binding-deficient PITP transgene
Escherichia coli strain DY380 was transformed with P1 clone 4232 (a gift from Bruce Hamilton, University of California at San Diego), which contains the entire PITP genomic locus in a P1 artificial chromosome (PAC) vector harboring a kanamycin resistance selection marker, to generate E. coli strain DY380-Tg(PITP). DY380 is permissive for homologous recombination when electroporated linear double-stranded DNA is introduced into this E. coli host (25, 26). The T59D missense substitution was incorporated into exon 3 of the PITP gene as follows. A pair of 100mer oligonucleotides precisely complementary to each other in their respective 3' 20 nucleotides was constructed. The 20 bp complementary regions spanned the threonine 59 codon, which was converted during oligonucleotide synthesis from the wild-type ACA nucleotide sequence to GAG (and the corresponding reverse complement). Oligonucleotides were annealed at 42°C for 1 h, and the remaining 3' recessed ends were filled in with Klenow fragment of DNA polymerase I (Promega, Madison, WI). The resultant 200 bp linear double-stranded DNA fragment represented the targeting cassette for incorporating the T59D missense mutation into exon 3 of the PITP structural gene.

The 200 bp target fragment was electroporated into DY380-Tg(PITP) cells prechallenged at 42°C for 15 min (to induce the recombineering machinery), transformants were selected in Luria broth supplemented with kanamycin (50 µg/ml), survivors were serially diluted into five 96-well culture plates to a density of 10 colony-forming units per well, and the individual pools were expanded. Aliquots from each well were collected and corresponding DNA templates were prepared for screening using a mismatch amplification mutation assay (MAMA) PCR. MAMA-PCR used a forward primer (5'-GGCGAGAAAGGCGAGTACGAG-3') specific for the mutant T59D codon (underlined) and reverse primer (5'-CTCACATGCCACACACTCC-3') that hybridizes to sequences within the PITP gene not represented in the original 200 bp targeting fragment. Aliquots yielding a 300 bp PCR product diagnostic of Pitp^T59D clones identified the wells of interest, and the corresponding well cultures were subjected to limit dilution and plated for single colonies. Individual colonies derived from well pools of interest were rescreened by MAMA-PCR. Positive clones yielded candidate purified P1 Tg(Pitp^T59D clones. Identities were verified by nucleotide sequence analysis of PITP exon 3 of individual candidates. We estimate a 1:12,000 ratio of Tg(Pitp^T59D) to Tg(PITP) in the final pools.

Immunoblotting and ELISA
Whole mouse brains or duodenal sections were solubilized in RIPA buffer (50 mM Tris-HCl, pH 7.2, 1.0% Nonidet P-40, 0.1% SDS, 0.15 mM NaCl, and 1x protease inhibitor cocktail tablet; Roche, Indianapolis, IN). Protein was estimated using BCA (Pierce, Rockford, IL) for purposes of normalization in immunoblot and ELISA experiments. For immunoblotting experiments, 75 µg of brain lysate was resolved by SDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). Membranes were subsequently decorated with PITP-specific antibody as described previously (20), and with a ß-actin specific antibody (Oncogene, Carpinteria, CA) for purposes of normalization. For ELISA analyses, serial amounts of brain lysate protein for each experimental sample (1 µg, 100 ng, and 10 ng) were coated, in duplicate, on 96-well plastic plates. Samples were dried overnight, washed in 0.5% Tween 20, blocked with 2% BSA, incubated with the appropriate primary antibody overnight at room temperature, washed in PBS, incubated with secondary antibody, and finally developed using o-phenylenediamine reagent (Sigma, St. Louis, MO).

Blood chemistry
Whole blood was collected from mice immediately after decapitation or via heart puncture (in 2 mM EDTA, pH 7.2, final concentration for plasma), clotted, and clarified by low-speed centrifugation. Serum glucose was determined by the Trinder assay (Sigma). Plasma triglyceride (TG) measurements were performed on 30 µl plasma aliquots at the Animal Clinical Chemistry and Gene Expression Laboratories at the University of North Carolina-Chapel Hill. Agarose electrophoresis of whole serum used the Titan Gel Lipoprotein Kit (Helena Laboratories, Beaumont, TX) according to the manufacturer's specifications. Resolved samples were developed by staining of gels in 0.1% Fat Red 7B (w/v) in 95% methanol. Chylomicron densities were determined by scanning dried Titan gels using a Bio-Rad VersaDoc imaging system (525 nm) and quantified by the Quantity One software package.

Whole carcass analyses
Carcass compositional analyses were performed as described (21). Briefly, eviscerated carcasses were weighed and body water content determined after drying. Dried carcasses were ground to homogeneity and extracted with petroleum ether in a Soxhlet apparatus to determine fat mass and fat-free dry mass. Carcass ash was measured after limit incineration of fat-free dry mass material.

Histological methods
Mice were prepared for all analyses by anesthesia with 1.25% Avertin (Sigma) and fixed by heart perfusion with sequential phosphate-buffered saline and 4% paraformaldehyde solutions. Intestines, brains, and livers were harvested, intestines were flushed with fixative, and all tissues were postfixed in 4% paraformaldehyde for 24 h. When dehydration was required, tissues were embedded in paraffin, and 5 µm thick sections were cut and mounted onto slides. When frozen samples were required, tissues were embedded in OCT, and 12 µm thick sections were cut and mounted.

For intestinal and liver staining with osmium (27), paraffin sections were rehydrated in a step series starting with xylene and terminating with 50% ethanol (EtOH) (i.e., xylene, 95% EtOH, 90% EtOH, 80% EtOH, 70% EtOH, 60% EtOH, and 50% EtOH), stained for total lipid by incubation with 5% potassium dichromate and 2% OsO₄ for 8 h, and counterstained with toluidine blue O. Samples were then dehydrated in a 50% EtOH-to-xylene series that was the reverse of the rehydration series described above. Dehydrated sections were mounted in Permount (Fisher Scientific, Atlanta, GA). Oil Red O staining was used to identify neutral lipid (27). Frozen sections mounted onto slides were incubated in 85% propylene glycol, stained with Oil Red O, counterstained with hematoxylin, and mounted in glycerol.

For whole brain analyses, fixed samples were washed for several days in phosphate-buffered saline. Brains were then sliced in half along the sagittal plane and embedded in paraffin, and 5 µm-thick sections were cut. Sections were rehydrated as described above and incubated with anti-glial fibrillary acidic protein (GFAP) antibody (EMD Biosciences CalBiochem, San Diego, CA). Decorated slices were developed with the Vectastain ABC kit (Vector Laboratories, Burlingame, CA), followed by counterstaining with toluidine blue. Samples were finally dehydrated in xylene/EtOH (50%) and mounted in Permount (Fisher Scientific).

-Tocopherol measurements
Whole brains were harvested from decapitated mice, and -tocopherol was extracted with chloroform as described (21). Samples were resolved by HPLC using a Perkin-Elmer Life Sciences model LC200 gradient pump with an AS 200 Autosampler and an LC 295 ultraviolet-visible light detector.

Liver glycogen analysis
Whole livers were extracted from euthanized animals and homogenized, and glycogen was recovered by acid extraction (28). Recovered material was quantitatively hydrolyzed to glucose by amyloglucosidase, and released glucose was quantified by Trinder assay (Sigma).

Hippocampal field excitatory postsynaptic potential recordings
Age-matched (P4–P6) PITP^+/+ and Pitp^0/0 mice were anesthetized with an overdose of pentobarbital barbiturate and decapitated upon the disappearance of corneal reflexes, in compliance with U. S. Department of Health and Human Services and University of North Carolina guidelines. Brains were dissected immediately, and dorsal hippocampi were cut in 400 µm coronal slices in ice-cold oxygenated dissection buffer (75 mM sucrose, 87 mM NaCl, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 26 mM NaHCO₃, 10 mM glucose, 7 mM MgCl₂, 0.5 mM CaCl₂, and 1.3 mM ascorbic acid). Hippocampal slices were equilibrated for 30 min in a submersion chamber at 35°C filled with artificial cerebrospinal fluid (124 mM NaCl, 3 mM KCl, 1.25 mM Na₂PO₄, 26 mM NaHCO₃, 1 mM MgCl₂, 2 mM CaCl₂, and 20 mM D-glucose, saturated with 95% O₂ and 5% CO₂; 315 mOsm and pH 7.25) and then held at room temperature until use. For recordings, hippocampal slices were placed in an interface chamber, maintained at 31°C, and perfused with oxygenated artificial cerebrospinal fluid. Synaptic responses were evoked by stimulating the Schaffer collaterals with a bipolar stimulating electrode (FHC, Inc., Bowdoin, ME), and field excitatory postsynaptic potentials (fEPSPs) were recorded in stratum radiatum of the CA1 region. Input-output curves were generated by systematically varying the stimulation intensity and measuring the fEPSP slope. Fiber volley amplitude was compared with the fEPSP slope to relate presynaptic to postsynaptic responses. The paired-pulse facilitation of the fEPSP slope and amplitude was examined by adjusting the stimulation intensity to the half-maximal response and systematically varying the interstimulus interval between two stimulations. Statistical analyses used a multivariate two-way ANOVA, with significance placed at P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
A murine strain that exclusively expresses a PtdIns binding-deficient PITP
PITP binds/exchanges PtdIns and PtdCho monomers between membrane bilayers in vitro. It is not yet established what each individual activity contributes to PITP action in cells. The availability of mutant PITP derivatives with selective defects in PtdIns binding (29), and the ability to generate PITP nullizygous (Pitp^0/0) mice with outstanding phenotypes (21), permit experimental assay of the contribution of PtdIns binding to PITP function in the mammal. To this end, the mutant Pitp^T59D was used to assess the in vivo consequences of inactivating PITP PtdIns binding activity. Residue threonine 59 is required for coordination of the inositol headgroup of PtdIns, and the introduction of any residue bulkier than threonine at this position evokes a selective steric incompatibility with PtdIns binding (Fig. 1 ) (10, 29, 30). Because choline is less bulky than inositol, PtdCho binding is unaffected in Pitp^T59D.

View larger version (47K): [in this window] [in a new window]   Fig. 1. Residue threonine 59 (T59) and phosphatidylinositol transfer protein (PITP)-mediated phosphatidylinositol (PtdIns) binding/exchange activity. Left panel: The van der Waals space of PITP residue threonine 59 is depicted in mesh mode (violet), whereas the van der Waals space of the inositol ring of bound PtdIns is rendered in dot mode (red and yellow). Note how closely the threonine 59 side chain abuts inositol ring space. Right panel: Same as the left panel except an aspartate side chain is modeled for threonine at PITP residue 59 to depict the T59D missense mutation. The aspartate 59 van der Waals space is rendered in green mesh. Note the steric incompatibility between aspartate 59 and the inositol ring of PITP-bound PtdIns. Both PITP and PitpT59D images were generated with PyMOL 0.99 using the PtdIns-bound PITP crystal structure as a framework (Protein Data Bank accession number 1UW5) (30).

PITP

Tg(PITP)

Tg(PITP)

Pitp

Tg(PITP)

Pitp

Pitp

;Tg(PITP)

PITP

Tg(PITP)

The PITP PAC mutagenesis scheme is outlined in Fig. 2A . The Pitp^T59D PAC [referred to as Tg(Pitp^T59D)] was injected into C57BL/6JxC3H hybrid pronuclei to generate founder mice. Standard mating strategies were used to generate PITP^+/0;Tg(Pitp^T59D) parental lines from which Pitp^0/0;Tg(Pitp^T59D) progeny were derived. The presence of Tg(Pitp^T59D) was diagnosed by probing for DNA sequences unique to the bacterial plasmid backbone of this vector (Fig. 2B) (see Materials and Methods). However, because an intact 76 kDa PITP locus was used as the mutagenesis substrate, diagnostic assays for Tg(Pitp^T59D) were more complicated. Additional methods were required to independently confirm mouse genotypes (see below). To that end, a robust MAMA-PCR strategy that distinguishes the endogenous PITP locus from ectopic Tg(Pitp^T59D) was developed for purposes of diagnostic genotyping of transgenic mice (see Materials and Methods) (Fig. 2B). Demonstration that this assay reliably detects Pitp^T59D in genomic DNA preparations is shown in Fig. 2C.

View larger version (31K): [in this window] [in a new window]   Fig. 2. Generation of mice expressing a PtdIns binding-deficient PITP. A: P1 artificial chromosome (PAC) mutagenesis strategy. Tg(PITP+) represents P1 clone 4232, which contains the entire 76 kb PITP+ structural gene. The PitpT59D missense substitution was introduced into this PAC by recombineering-driven site-directed mutagenesis, as described in Materials and Methods. dNTP, deoxynucleoside triphosphate. B: Diagnosis of Tg(PitpT59D) in mouse genomic DNA. The mutant transgene was detected in candidate transgenic mouse founder lines using a PCR strategy screening for the presence of the PAC vector backbone. Arrows indicate the positions of forward and reverse diagnostic PCR primers that yield a 350 bp product detected exclusively in genomic DNA prepared from founder mice carrying Tg(PitpT59D). C: Confirmation of Tg(PitpT59D) in candidate transgenic founder lines. Arrows indicate primers used in mismatch amplification mutation assay (MAMA)-PCR analysis (which yields a 300 bp PCR product; left panel) to specifically reveal the PitpT59D allele. D: Tg(PitpT59D) is actively transcribed. The upper panel illustrates the RT-PCR strategy used to detect transcripts in Tg(PitpT59D) mice. Solid arrows indicate MAMA-PCR primers used to distinguish endogenous PITP+ mRNA (WT59-5) transcripts from those of PitpT59D (TD59-5). In both cases, the diagnostic PCR products are 350 bp. Dashed arrows indicate the forward and reverse PCR primers (PITP-5 and PITP-3) used in RT-PCR assays to reveal full-length PITP transcripts (820 bp product) without distinguishing PITP+ transcripts from those of PitpT59D. The lower panel demonstrates this method. Mouse genotype is indicated at the top and RT-PCR products are identified at the bottom of individual lanes. E: Confirmation that PitpT59D is properly expressed. Nucleotide sequence analysis of the RT-PCR product generated by the PITP-5/PITP-3 primer combination. The left panel shows the cDNA sequence readout of relevant codons from a PITP+/+ mouse. The middle and right panels are the corresponding readouts from PITP+/0,Tg(PitpT59D) and Pitp0/0,Tg(PitpT59D) mice, respectively. Genotypes are indicated at the top, and relevant sequences are identified at the top of the red boxes. Corresponding amino acids are listed: Asp, aspartate; Thr, threonine. Note that the PITP+/0,Tg(PitpT59D) reactions reveal sequences derived from both PITP+ from PitpT59D alleles.

Tg(Pitp

Tg(Pitp

Tg(Pitp

PITP

PITP

;Tg(Pitp

PITP

PITP

Pitp

Tg(Pitp

PITP

PtdIns binding/transfer is required for PITP function in mammals
Standard matings between PITP^+/0;Tg(Pitp^T59D) males and females generated the full complement of expected genotypes. As all PITP-sufficient progeny exhibited phenotypes indistinguishable from the wild type, irrespective of their Tg(Pitp^T59D) genotype, we restricted our attention to comparisons of PITP^+/+;Tg(Pitp^T59D), Pitp^0/0, and Pitp^0/0;Tg(Pitp^T59D) littermates. Lifespan-monitoring experiments demonstrated that Tg(Pitp^T59D) expression does not rescue the neonatal and perinatal lethality of otherwise PITP nullizygous offspring. For Pitp^0/0;Tg(Pitp^T59D) progeny, the rapid onset of postnatal mortality characteristic of PITP nullizygous mice was on display (Fig. 3A ). Comparison of Pitp^0/0 and Pitp^0/0;Tg(Pitp^T59D) progeny at P3 and P6 revealed that both strains exhibited the wasting phenotype typical of PITP nullizygotes. This wasting was associated with dramatic reductions in the contribution of subcutaneous fat to total body mass (data not shown). Additional analyses of Pitp^0/0;Tg(Pitp^T59D) mice demonstrated that these also exhibited the striking hypoglycemia and intestinal and liver microvesicular steatosis disorders that accompany PITP nullizygosity (Table 1 ). Phenotypic manifestations of the robust neurodegenerative disease characteristic of Pitp^0/0 neonates were also apparent (data not shown). In sum, the Pitp^0/0 and Pitp^0/0;Tg(Pitp^T59D) phenotypes were indistinguishable based on criteria of severity and rate of onset.

View larger version (43K): [in this window] [in a new window]   Fig. 3. PitpT59D is not a biologically functional protein. A: Lifespans for PITP+/+ (open circles), Pitp0/0 (closed circles), and Pitp0/0,Tg(PitpT59D) (open squares) mice are plotted. The data were obtained from a dedicated pool of 176 mice with a genotypic distribution of 118 PITP+/+, 26 Pitp0/0, and 32 Pitp0/0,Tg(PitpT59D). B: Images of PITP+/+, Pitp0/0, and Pitp0/0,Tg(PitpT59D) mice taken at the indicated ages. C: Tg(PitpT59D) protein is expressed at physiological levels. Immunoblot analysis of PITP immunoreactive species from whole brain (WBr), duodenum (Dnm), and liver, collected from P6 littermates of the indicated genotypes, using a PITP-specific C-terminal polyclonal antibody (top panels). The bottom panels depict the corresponding blots that were stripped and reprobed with a ß-actin-specific antibody for purposes of normalization.

View this table: [in this window] [in a new window]   TABLE 1. PitpT59D is nonfunctional in the mouse

Tg(Pitp

Pitp

Pitp

;Tg(Pitp

PITP

PITP

Pitp

Pitp

;Tg(Pitp

An allelic series for titrating PITP expression in mice
PITP-deficient mice elaborate multiple phenotypes. Manifestation of some of these phenotypes is sensitive to amounts of PITP protein (21, 22). These findings suggest that titration of PITP function in mice is a viable approach for discerning relationships between the complex phenotypes that accompany PITP insufficiencies. To this end, we took advantage of the availability of Pitp⁰ and Pitp^vb alleles to create a series of murine strains with graded levels of PITP expression. The Pitp^vb allele reduces the expression of wild-type PITP as a result of the insertion of an IAP element into intronic sequences of the PITP structural gene (22). Because the Pitp⁰ allele was originally fixed in the 129SVJ genetic background, and the hypomorphic Pitp^vb phenotype is sensitive to genetic modifiers (22), Pitp⁰ was incorporated into the C57BL/6J genetic background by serial backcross (see Materials and Methods). The C57BL/6J background elicits the most severe phenotype for the Pitp^vb homozygote mice and is minimized for the Pitp^vb genetic modifiers detected in other murine strain backgrounds (22). Comparisons of the morbidity of Pitp^0/0 offspring as a function of genetic background (C57BL/6J vs. 129SVJ) demonstrated that the lifespans of nullizygotes were very similar in both genetic background contexts (50% mortality at 4.2 ± 1.2 vs. 4.8 ± 0.7 days, respectively).

Standard mating schemes were used to generate C57BL/6J-derived mice harboring Pitp^+/+, Pitp^+/0, Pitp^vb/vb, Pitp^vb/0, and Pitp^0/0 allelic combinations. Relative levels of endogenous PITP in whole brain cytosol extracts were prepared from mice of each genotype and compared by immunoblotting and quantitative ELISA (Fig. 4A , B). Again, in both experiments, antibodies specific for PITP were used to avoid confounding issues presented by the highly similar PITPß isoform in these mice. In that regard, PITP^+/+, PITP^+/vb, PITP^+/0, Pitp^vb/vb, Pitp^vb/0, and Pitp^0/0 mice express 100, 94, 61, 18, 13, and <3% of wild-type PITP levels, respectively (Fig. 4B). These data indicate that the titration range for PITP was a satisfactorily broad one. For purposes of these studies, we focus on PITP^+/+ and Pitp^0/0 strains as positive and null controls, respectively, and Pitp^vb/vb and Pitp^vb/0 mice as experimental variables.

View larger version (23K): [in this window] [in a new window]   Fig. 4. Quantification of PITP protein levels in a series of mice harboring various PITP mutant allele combinations. A: Immunoblot analyses of PITP immunoreactive species from whole brain lysates collected from P8 mice of the indicated genotypes. The top panel presents immunoblot profiles obtained by probing the indicated lysate preparations with a PITP-specific antibody. The bottom panel represents the same blot stripped and reprobed with a ß-actin-specific antibody as a normalization control. B: ELISA analyses of PITP protein levels from the same whole brain lysate preparations described for A. Results are expressed as PITP levels relative to PITP+/+ controls (set at 100%) after correction for relative ß-actin ELISA values. The values represent averages (plus SD) from triplicate measurements for each of three independent brain samples collected from three mice of each genotype.

Phenotypes of mice with graded reductions in PITP expression

Pitp

Pitp

Pitp

PITP

PITP

PITP

Pitp

Pitp

Pitp

Pitp

Pitp

PITP

Pitp

PITP

Pitp

Pitp

Pitp

View larger version (35K): [in this window] [in a new window]   Fig. 5. Phenotyping of mice with graded levels of PITP protein. A: Lifespan plots for PITP+/+ (open circles), Pitpvb/vb (closed squares), Pitpvb/0 (open squares), and Pitp0/0 (closed circles) are shown. B: Average body masses of PITP+/+ (open circles), Pitpvb/vb (closed squares), Pitpvb/0 (open squares), and Pitp0/0 (closed circles) neonates are plotted in the P5 through P21 age window. Values represent averages over lifespan for progeny derived from four independent litters for each genotype. No Pitp0/0 neonates survived past P9 in these dedicated pools of animals. C: Images of P9, P14, and P19 PITP+/+ and Pitpvb/0 mice (left and right animals, respectively, in each panel).

Quantitative relationship between PITP activity and steatosis of intestine and liver

Pitp

Pitp

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View larger version (59K): [in this window] [in a new window]   Fig. 6. Chylomicron metabolism in mice with varying levels of PITP activity. A, B: Osmium staining of intestinal (A) and liver (B) tissue. Panels depict paraffin-embedded, osmium-stained intestinal and liver sections of P8 mice from the indicated genotypes. Steatosis is scored by intensity of osmium staining (black). C: Postprandial plasma triglyceride (TG) levels as a function of age. Plasma was harvested from three age-matched PITP+/+ (open circles), Pitp0/0 (closed circles), Pitpvb/0 (open squares), and Pitpvb/vb (closed squares) animals and measured in triplicate for each mouse at each time point. D: Brain -tocopherol measurements. Whole brains from age-matched (P6) PITP+/+ and Pitp0/0 mice, as well as from age-matched (P16) PITP+/+, Pitpvb/0, and Pitpvb/vb animals, were harvested and corresponding extracts were assayed for -tocopherol. Measurement averages with SD are shown. Genotypes are listed at bottom.

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Reconstitution of PITP in small intestine of Pitp^0/0 mice
The involvement of PITP in chylomicron trafficking from nullizygous enterocytes is puzzling, as the affected organelle [endoplasmic reticulum (ER)] is the major intracellular site of lipid synthesis. Moreover, it remains unknown whether this pathology reflects a tissue-autonomous defect and what CRD contributes to the complex disease phenotypes of Pitp^0/0 neonates. To this end, a transgene was engineered in which duodenum-specific expression of PITP cDNA is driven by the intestinal fatty acid binding protein enhancer [Tg(P_IFABP::PITP)] (Fig. 7A ) (see Materials and Methods). Immunoblotting experiments of brain and intestine culled from PITP^+/+, Pitp^0/0, and Pitp^0/0;Tg(P_IFABP::PITP) offspring confirmed that the transgene supports a satisfactory tissue-specific expression of physiologically relevant levels of PITP (Fig. 7A).

View larger version (66K): [in this window] [in a new window]   Fig. 7. Intestinal expression of PITP in Pitp0/0 mice. A: The upper panels describe the vectors used to generate Tg(PIFABP::PITP+). Plasmid pNSE-EX4 was used as an expression vector backbone, and PITP expression was engineered under the control of the intestinal fatty acid binding protein promoter (PIFABP) (see Materials and Methods). The lower panels show the results of immunoblot analyses using lysates prepared from whole brain (WBr) or duodenum (Dnm) preparations collected from mice of the indicated genotypes. Immunoblot profiles at top monitor PITP, whereas the immunoblot profiles at bottom record the corresponding ß-actin profiles for purposes of normalization. B: Age-matched (P6 littermates) duodenal sections stained with osmium. Genotypes are indicated. C: Liver sections from P6 littermates stained with osmium. Genotypes are indicated.

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View larger version (28K): [in this window] [in a new window]   Fig. 8. Chemical analyses of Pitp0/0 mice expressing PITP in the duodenum. A: Postprandial serum chylomicron levels. A Titan lipoprotein agarose gel is shown at left. Genotypes of P5 littermates are indicated, and bands are identified as follows: Chyl, chylomicron; LDL, low density lipoprotein; HDL, high density lipoprotein. The right panel represents quantification (by densitometry) of chylomicron bands from a total of five animals of each indicated genotype. Tg indicates the Tg(PIFABP::PITP+) allele. PITP+/+ values were uniformly set at 100%, and Pitp0/0 and Pitp0/0,Tg(PIFABP::PITP+) values were related to that benchmark. B: Postprandial plasma TG levels as a function of age. Plasma TGs from three age-matched littermates (as indicated) were measured. Avg. indicates the average of all sample measurements for that particular genotype [PITP+/+, open circles; Pitp0/0, closed circles; Pitp0/0,Tg(PIFABP::PITP+), open triangles]. C: Whole brain -tocopherol measurements. Four age-matched littermates of each indicated genotype were analyzed. Averaged values with SD are reported. Tg indicates Tg(PIFABP::PITP+).

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Lifespan and body mass measurements indicated that duodenal PITP expression levied modest improvement in these parameters relative to Pitp^0/0 siblings. Although 75% of the Pitp^0/0 offspring expired within the first 3 days after birth, some 80% of their Pitp^0/0;Tg(P_IFABP::PITP) siblings survived past this benchmark (Fig. 9A ). A 75% mortality standard was not reached for the Pitp^0/0;Tg(P_IFABP::PITP) neonates until 8 days after birth. Again, although modest, this represents an effective doubling of the Pitp^0/0 lifespan. Body mass measurements also showed measurable increases in weight gain differential for Pitp^0/0;Tg(P_IFABP::PITP) and Pitp^0/0 neonates (Fig. 9B). For instance, at P7, the respective mass differential was 3.1 ± 0.3 versus 2.3 ± 0.5 g (P = 0.026).

View larger version (29K): [in this window] [in a new window]   Fig. 9. Characterization of Pitp0/0,Tg(PIFABP::PITP+) mice. A: Lifespan profiles for PITP+/+ (open circles), Pitp0/0 (closed circles), and Pitp0/0,Tg(PIFABP::PITP+) (open triangles) are compared. The data were obtained from a dedicated pool of 148 mice with a genotypic distribution of 100 PITP+/+, 21 Pitp0/0, and 27 Pitp0/0,Tg(PIFABP::PITP+). B: Visual characterization and body mass measurements of Pitp0/0,Tg(PIFABP::PITP+) mice. The upper panel shows images of P4 (left) and P7 (right) mice. For each panel, genotypes of mice (from left to right) are PITP+/+, Pitp0/0, and Pitp0/0,Tg(PIFABP::PITP+). The lower panel charts body mass as a function of age for PITP+/+ (open circles), Pitp0/0 (closed circles), and Pitp0/0,Tg(PIFABP::PITP+) (open triangles). Data were obtained from the same dedicated pool of 148 mice used for A. Body mass values were averaged for each genotypic group. SD values are shown.

PITP expression and glucose homeostasis

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View larger version (12K): [in this window] [in a new window]   Fig. 10. PITP levels and glucose homeostasis. A: Average serum glucose values measured from age-matched (P4–P6) PITP+/+, Pitp0/0, Pitp0/0,Tg(PIFABP::PITP+), Pitpvb/0, and Pitpvb/vb mice. The genotypes are indicated at bottom. B: Serum glucose levels as a function of age. PITP+/+ (open circles), Pitp0/0 (closed circles), Pitp0/0,Tg(PIFABP::PITP+) (open triangles), Pitpvb/0 (open squares), and Pitpvb/vb (closed squares) serum glucose were determined at the indicated ages. No Pitp0/0 or Pitp0/0,Tg(PIFABP::PITP+) measurements were available beyond P8, as no animals survived past this threshold. Data were obtained from triplicate determinations from each of three mice of each genotype at each time point. The serum glucose values were quite constant for Pitp0/0 and Pitp0/0,Tg(PIFABP::PITP+) mice in the P3–P8 time window (2.3 ± 0.5 vs. 3.8 ± 0.6 mM, respectively), and these differences were significant (P < 0.0002).

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PITP expression and cerebellar neurodegeneration
A hallmark phenotype of Pitp^0/0 mice is a robust hindbrain and cerebellar inflammatory disease characterized by reactive gliosis in these tissues (21). This pathology is scored by staining for GFAP, a marker for activated astrocytes (Fig. 11A ). It is suggested that the Pitp^0/0 cerebellum is particularly sensitive to damage wrought by a systemic energy crisis, such as that precipitated by the CRD and hypoglycemia of Pitp^0/0 neonates (21). Consistent with this proposal, cerebellar inflammatory disease was substantially alleviated in both Pitp^vb/vb and Pitp^0/0;Tg(P_IFABP::PITP) neonates, as indicated by dramatic reductions in GFAP-positive cells in tissue derived from those mice. By contrast, hindbrain inflammation was not alleviated in either Pitp^0/0;Tg(P_IFABP::PITP) or Pitp^vb/0 neonates (Fig. 11B). The persistent hindbrain inflammation presumably reflects some intrinsic fragility/deficit in Pitp^0/0 hindbrain neurons.

View larger version (74K): [in this window] [in a new window]   Fig. 11. Cerebellar neurodegeneration and PITP expression. Sagittal sections of cerebellum (A) or hindbrain (B) were infiltrated with paraffin and stained for glial fibrillary acidic protein. Genotypes are indicated; Tg indicates the Tg(PIFABP::PITP+) allele. Tissue sections were from P8 animals except for Pitpvb/0 mice, for which P14 animals were used. These ages were chosen on the basis that neurodegenerative disease had progressed to the obvious whole body tremor stage.

PITP nullizygosity and efficacy of synaptic transmission

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View larger version (34K): [in this window] [in a new window]   Fig. 12. Synaptic transmission is not affected by PITP defects. A: PITP is highly expressed in the hippocampus. At bottom is a representative montage of two merged sections of a sagittal brain slice from a PITP+/Pitp::neo/puro/LacZ mouse (P10) stained for ß-galactosidase activity. ß-Galactosidase expression, driven from the endogenous PITP enhancer, is particularly robust in the hippocampus. The hippocampus region is shown at higher magnification in the top panel. B: Summary input-output curves of field excitatory postsynaptic potentials (fEPSPs) recorded in CA1 of the hippocampus and evoked by Schaffer collateral stimulation. The input-output relationship is similar in PITP+/+ and Pitp0/0 mice (open and closed circles, respectively). Sample sizes were n = 4 each for PITP+/+ and Pitp0/0 animals. The waveform depicts a sample input-output recording from a PITP+/+ mouse. C: Comparison of fiber volley amplitude to the fEPSP slope in PITP+/+ and Pitp0/0 mice (open and closed circles, respectively). The similar curves between the two groups suggest that the relationship between presynaptic release and postsynaptic response is unchanged in PITP nullizygous mice. Sample sizes were n = 4 each for PITP+/+ and Pitp0/0 animals. D: Paired-pulse facilitation of the fEPSP responses is similar in PITP+/+ and Pitp0/0 mice (open and closed circles, respectively), indicating that elimination of PITP activity fails to alter the probability of neurotransmitter release. The slope of the second response is plotted as a percentage of the slope of the first response. The waveform depicts a representative paired-pulse response recorded from a PITP+/+ mouse. Sample sizes were n = 5 each for PITP+/+ and Pitp0/0 mice. E: Same as D except that the fEPSP amplitude was measured instead of the slope. Sample sizes were n = 5 PITP+/+ and n = 6 Pitp0/0 mice, respectively.

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Presynaptic terminals of stratum radiatum neurons were also examined by quantitative morphometry. Defects in the synaptic vesicle cycle should result in abnormal synaptic vesicle densities in such presynaptic terminals. PITP^+/+ and Pitp^0/0 littermate mice (four each) were perfused with mixed aldehydes, and brains were collected and embedded. Embedded brains were sectioned through the hippocampus, further processed, and flat-embedded in plastic (34). Blindly coded sample blocks were prepared from the CA1 region of hippocampus, and morphometric analyses focused on synapses in the stratum radiatum. A total of 117 images of randomly selected terminals with recognizable synapses were collected, 59 from PITP^+/+ animals and 58 from Pitp^0/0 mice. For each terminal, vesicles were counted within a 0.2 µm square area: 0.1 µm lateral to each side from the center of the opposing postsynaptic density and 0.2 µm back into the terminal. The number of vesicles per examined area ranged from 0 to 19. The PITP^+/+ and Pitp^0/0 mice averaged 6.4 and 7.7 vesicles per synapse, respectively. Although there were the expected variations in the numbers of vesicles per synapse, no obvious correlation between vesicle number and PITP status was discerned. Thus, the collective electrophysiological and morphometric data indicate that the synaptic vesicle cycle of Pitp^0/0 mice is unperturbed, and that neurodegeneration is unlikely to be a consequence of deficits in synaptic transmission.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Here, we report a detailed analysis of PITP function in mice. These studies take advantage of transgenic approaches to achieve three purposes. First, the contribution of PtdIns binding to PITP function in vivo was evaluated. Second, a series of isogenic mouse strains with graded reductions in PITP activity was analyzed to examine the relationships between the complex pathologies apparent in Pitp^0/0 neonates. Third, the consequences of reconstituting PITP expression solely in the small intestine of Pitp^0/0 animals were assessed. The conclusions derived from these analyses are as follows: i) PtdIns binding activity is essential for PITP function in the mouse; ii) the lifespan of neonatal and juvenile mice is highly sensitive to levels of PITP; iii) large reductions in PITP expression are required for the elaboration of intestinal and liver steatotic phenotypes and hypoglycemia; iv) intestinal steatosis is a tissue-autonomous pathology that is rescued by tissue-specific expression of PITP; v) hindbrain neurodegenerative processes can be largely uncoupled from intestinal/liver steatosis and hypoglycemia, whereas cerebellar inflammatory disease cannot; and vi) elimination of PITP activity has no apparent consequence for synaptic efficacy, even in neurons that normally exhibit high levels of PITP expression. Together, these data provide the first in vivo demonstration of a meaningful biological role for PtdIns binding by PITP. The data also bolster the idea that PITP plays a role in the biogenesis and transport of lipid/lipoprotein cargo from intracellular organelles.

PtdIns binding and PITP function
Current models suggest that PITP is devoted primarily to phosphoinositide synthesis, particularly PtdIns-4,5-P₂ synthesis (35–38). Such a role could involve a direct coupling between PITP and PtdIns 4-OH kinases, in which PITP presents PtdIns to the enzyme and thereby potentiates its catalytic activity. A more indirect PtdIns delivery mechanism, in which PITP supplies PtdIns to sites of active signaling from other intracellular membranes (e.g., by PtdIns transfer), is also plausible. Alternatively, PITP could stimulate PtdIns-4,5-P₂ synthesis by potentiating phospholipase D-mediated hydrolysis of PtdCho [and subsequently stimulating PtdIns-4-phosphate 5-OH kinase activity via the production of the phosphatidic acid hydrolytic product (39)]. The former two models predict a critical role for PtdIns binding by PITP, whereas the latter model emphasizes PtdCho binding. The finding that physiological levels of expression of Pitp^T59D, as the sole source of PITP in the mouse, recapitulate phenotypes of Pitp^0/0 mice, identifies PtdIns binding as an essential functional property of PITP. This demonstration is most consistent with either PtdIns presentation, or PtdIns transfer, mechanisms for PITP function in vivo. To distinguish between the presentation and transfer mechanisms, it is necessary to assess the in vivo functionality of mutant PITP forms with defects in PtdCho binding. Unfortunately, available structural information does not reveal how to generate such PtdCho binding-defective forms of PITP.

Phenotypic consequences of graded reductions in PITP expression
Pitp^0/0 mice experience a complex disease course of intestinal and hepatic steatosis, hypoglycemia, and inefficient glycogenolysis (stemming from pancreatic insufficiency) and a fulminating spinocerebellar neurodegenerative disease. The neurodegeneration is marked by reactive gliosis of the cerebellum and hindbrain and aponecrotic pathologies in the spinal cord (21). The relationships between these various pathologies is not obvious, however. Analyses of isogenic mouse lines with graded reductions in PITP expression clarify some of these relationships. First, it is clear that the hindbrain inflammatory and spinal cord neurodegenerative diseases are intrinsic pathologies of PITP-insufficient mice, as these can be uncoupled from intestinal/liver steatosis and hypoglycemia. This is most apparent in the case of Pitp^vb/vb mice, which express 20% of wild-type levels of PITP activity. Pitp^vb/vb animals suffer from both hindbrain and spinal cord neurodegeneration (22, 31) but fail to exhibit any symptoms of overt intestinal/liver steatosis or hypoglycemia. By contrast, both intestinal/liver steatosis and hypoglycemia are apparent only in mice in which PITP activity is reduced by 90% (i.e., Pitp^0/0 and Pitp^vb/0 lines). Moreover, the severities of the respective pathologies are closely correlated. Pitp^vb/0 mice exhibit a less severe intestinal/liver steatosis than Pitp^0/0 siblings. Pitp^vb/0 mice also show a significantly reduced hypoglycemia. These data are consistent with the view that the hypoglycemia of PITP-insufficient mice is secondary to intestinal/hepatic lipid transport defects.

Reconstitution of PITP expression in small intestine
The intestinal steatosis of Pitp^0/0 mice is an intriguing phenotype. Its characteristics are consistent with a defect in the biogenesis/transport of chylomicrons from the enterocyte ER (21). Indeed, the steatotic condition (and its consequences) resemble those of human CRD (40, 41). One underlying mechanism for human CRD stems from defects in a specific isoform of the Sar1-GTPase (42). This raises the attractive possibility that specific components of the COPII ER vesicle-budding machinery are dedicated to the biogenesis of vesicles containing atypical cargo, such as chylomicrons.

A role for PITP in chylomicron biogenesis/trafficking is unanticipated, as it is not obvious why such a protein would be required for ER transport processes, given that the ER is the major site of lipid synthesis in cells. Moreover, the ER is not an acknowledged site of phosphoinositide signaling. Yet, the available data indicate a connection between PITP function and chylomicron metabolism. Specific defects in PITP PtdIns binding are sufficient to elicit CRD-like symptoms in Pitp^T59D-expressing mice. Moreover, reexpression of PITP in the small intestine of Pitp^0/0 mice alleviates the steatotic phenotype of this organ and partially rescues the biochemical defects associated with CRD. Pitp^0/0;Tg(P_IFABP::PITP) neonates also exhibit increased plasma TG levels, increases in the quantities of circulating chylomicrons, and increased brain -tocopherol levels (a vitamin whose transport into the circulation is dependent on the chylomicron pathway) relative to Pitp^0/0 controls. The rescue experiment additionally identifies the intestinal steatosis as a tissue-autonomous pathology in Pitp^0/0 mice.

Although reconstitution of PITP expression in the small intestine does have discernible effects, it nonetheless supports only a modest rescue of plasma TG deficits and hypoglycemia and only modest increases in the lifespan of transgenic Pitp^0/0 animals. Exacerbated liver steatosis is observed in these mice, however. As chylomicron remnants are taken up by the liver, remodeled, and reexported as VLDL particles, we suggest that defects in lipoprotein processing/biogenesis in Pitp^0/0 liver significantly neutralize the benefits realized by rescue of the intestinal steatosis. These data suggest a role for PITP in the hepatic lipoprotein biogenesis/transport pathway, a reasonable possibility given that the pathways for lipoprotein biogenesis and transport are similar in the small intestine and the liver (43–45). We predict that reconstitution of PITP expression in both the small intestine and the liver of a Pitp^0/0 mouse will yield what is a symptomatically Pitp^vb/vb mouse.

Differential causes of neurodegenerative disease
It was previously proposed that neonatal Pitp^0/0 cerebellum is especially susceptible to injury because its program of robust cell proliferation is incompatible with the hypoglycemic environment presented by the nullizygous mouse (21). The results reported here are consistent with this idea. Both Pitp^vb/0 and Pitp^0/0;Tg(P_IFABP::PITP) neonates are substantially free of the dramatic reactive gliosis that characterizes their Pitp^0/0 siblings. This is a telling result in the context of the Pitp^0/0;Tg(P_IFABP::PITP) mouse, as the cerebellum fails to express PITP yet is spared the normal course of inflammatory disease by PITP expression in the small intestine. Consistent with these data, cerebellar granule cells cultured from PITP^+/+ and Pitp^0/0 neonates show no obvious differences in vigor when cultured in vitro (i.e., under culture conditions in which defects associated with systemic lipid transport and hypoglycemia are irrelevant; data not shown).

As hindbrain and spinal cord defects are not alleviated by PITP expression in the duodenum, these pathologies reflect intrinsic neuronal defects of PITP-insufficient mice. These pathologies do not stem from deficiencies in the synaptic vesicle cycle, given that synaptic function is not adversely affected by genetic ablation of PITP activity in hippocampal neurons. Whether these intrinsic defects reflect improper neuronal development and defects in proper path finding (46) or some other signaling defect(s) remains to be determined.

	ACKNOWLEDGMENTS

 
This work was supported by National Institutes of Health Grant NS-37723 (to V.A.B.). B.D.P. was supported by the Whitehall Foundation. The authors thank G. Grossman, H. Mekheel, and J. de Marchena for valuable technical assistance and Margaret Ryan for help with Fig. 1. The authors also acknowledge the services of the University of North Carolina Lineberger Comprehensive Cancer Center Genome Analysis and Nucleic Acids Core Facilities, and Tammy Elliott and Melinda Beck from the University of North Carolina Clinical Nutrition Research Center (supported by National Institutes of Health Grant DK-56350). Particular thanks are extended to Kris Phend and Richard Weinberg (University of North Carolina) for quantitative morphometry data and to JrGang Cheng of the University of North Carolina Neuroscience Center Bacterial Artificial Chromosome Engineering Core Facility for reagents and helpful advice.

Manuscript received March 23, 2007 and in revised form May 11, 2007.

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