Paola Podini

Macropinocytosis: regulated coordination of endocytic and exocytic membrane traffic events

Macropinocytosis, a form of bulk uptake of fluid and solid cargo into cytoplasmic vacuoles, called macropinosomes, has been studied mostly in relation to antigen presentation. Early membrane traffic events occurring in this process are, however, largely unknown. Using human dendritic cells we show that a marked increase in the rate of macropinocytosis occurs a few minutes after application of two markers (small latex beads or dextran), depends on a slow intracellular Ca2+ concentration ([Ca2+]i) rise that precedes the PI3K-dependent step, and is preceded and accompanied by exocytosis of enlargeosomes compensating in part for the macropinocytic plasma membrane internalization. Unexpectedly, macropinosomes themselves, which share markers with endosomes, undergo Ca2+-dependent exocytosis so that, after ∼20 minutes of continuous bead or dextran uptake, an equilibrium is reached preventing cells from overloading themselves with the organelles. Large [Ca2+]i increases induced by ionomycin trigger rapid (<1 minute) exocytic regurgitation of all macropinosomes, whereas endosomes remain apparently unaffected. We conclude that, in dendritic cells, the rate of macropinocytosis is not constant but increases in a regulated fashion, as previously shown in other cell types. Moreover, macropinosomes are not simple containers that funnel cargo to an endocytic pathway, but unique organelles, distinct from endosomes by their competence for regulated exocytosis and other membrane properties.

Distribution and signaling of TREM2/DAP12, the receptor system mutated in human polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy dementia

Together with its adaptor protein, the adaptor protein of 12 kDa also known as KARAP and TYROBP (DAP12), triggering r (TREM2) is a stimulatory membrane receptor of the immunoglobulin/lectin‐like superfamily, well known in myeloid cells. In humans, however, loss‐of‐function mutations of TREM2/DAP12 leave myeloid cells unaffected but induce an autosomal recessive disease characterized, together with bone cysts, by a spectrum of pathological lesions in the cortex, thalamus and basal ganglia with clinical symptoms of progressive dementia (polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy). Nothing was known about the role of TREM2/DAP12 in brain cell biology and physiology. By confocal immunocytochemistry we demonstrate that, in both human and mouse cerebral cortex, TREM2/DAP12, strongly expressed by microglia, is also present in a fraction of neurons but not in astrocytes and oligodendrocytes. In contrast, in the hippocampal cortex TREM2‐expressing neurons are rare. Both in neurons and microglia the receptor appears to be located mostly intracellularly in a discrete compartment(s) partially coinciding with (or adjacent to) the Golgi complex/trans‐Golgi network. Four nerve cell lines were identified as expressing the intracellular receptor system. In living human microglia CHME‐5 and glioblastoma T98G cells, activation of TREM2 by its specific antibody induced [Ca2+]i responses, documenting its surface expression and functioning. Surface expression of TREM2, low in resting CHME‐5 and T98G cells, increases significantly and transiently (60 min) when cells are stimulated by ionomycin, as revealed by both surface biotinylation and surface immunolabeling. Our results provide the first information about the expression, distribution (mostly intracellular) and functioning of TREM2/DAP12 system in nerve cells, a necessary step in the understanding of the cellular mechanisms affected in polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy.

Alpha-lipoic acid prevents mitochondrial damage and neurotoxicity in experimental chemotherapy neuropathy

The study investigates if alpha-lipoic acid is neuroprotective against chemotherapy induced neurotoxicity, if mitochondrial damage plays a critical role in toxic neurodegenerative cascade, and if neuroprotective effects of alpha-lipoic acid depend on mitochondria protection. We used an in vitro model of chemotherapy induced peripheral neuropathy that closely mimic the in vivo condition by exposing primary cultures of dorsal root ganglion (DRG) sensory neurons to paclitaxel and cisplatin, two widely used and highly effective chemotherapeutic drugs. This approach allowed investigating the efficacy of alpha-lipoic acid in preventing axonal damage and apoptosis and the function and ultrastructural morphology of mitochondria after exposure to toxic agents and alpha-lipoic acid. Our results demonstrate that both cisplatin and paclitaxel cause early mitochondrial impairment with loss of membrane potential and induction of autophagic vacuoles in neurons. Alpha-lipoic acid exerts neuroprotective effects against chemotherapy induced neurotoxicity in sensory neurons: it rescues the mitochondrial toxicity and induces the expression of frataxin, an essential mitochondrial protein with anti-oxidant and chaperone properties. In conclusion mitochondrial toxicity is an early common event both in paclitaxel and cisplatin induced neurotoxicity. Alpha-lipoic acid protects sensory neurons through its anti-oxidant and mitochondrial regulatory functions, possibly inducing the expression of frataxin. These findings suggest that alpha-lipoic acid might reduce the risk of developing peripheral nerve toxicity in patients undergoing chemotherapy and encourage further confirmatory clinical trials.

Purkinje neuron Ca2+ influx reduction rescues ataxia in SCA28 model

Spinocerebellar ataxia type 28 (SCA28) is a neurodegenerative disease caused by mutations of the mitochondrial protease AFG3L2. The SCA28 mouse model, which is haploinsufficient for Afg3l2, exhibits a progressive decline in motor function and displays dark degeneration of Purkinje cells (PC-DCD) of mitochondrial origin. Here, we determined that mitochondria in cultured Afg3l2-deficient PCs ineffectively buffer evoked Ca²⁺ peaks, resulting in enhanced cytoplasmic Ca²⁺ concentrations, which subsequently triggers PC-DCD. This Ca²⁺-handling defect is the result of negative synergism between mitochondrial depolarization and altered organelle trafficking to PC dendrites in Afg3l2-mutant cells. In SCA28 mice, partial genetic silencing of the metabotropic glutamate receptor mGluR1 decreased Ca²⁺ influx in PCs and reversed the ataxic phenotype. Moreover, administration of the β-lactam antibiotic ceftriaxone, which promotes synaptic glutamate clearance, thereby reducing Ca²⁺ influx, improved ataxia-associated phenotypes in SCA28 mice when given either prior to or after symptom onset. Together, the results of this study indicate that ineffective mitochondrial Ca²⁺ handling in PCs underlies SCA28 pathogenesis and suggest that strategies that lower glutamate stimulation of PCs should be further explored as a potential treatment for SCA28 patients.

The Rest Repression of the Neurosecretory Phenotype Is Negatively Modulated by BHC80, a Protein of the BRAF/HDAC Complex

Expression of neurosecretion by nerve cells requires the levels of the transcription repressor element-1 silencing transcription factor (REST) to be very low. However, when high-REST clones of PC12 cells, defective of neurosecretion, were fused to other high-REST, non-neurosecretory cells, some neurosecretion was recovered. To clarify the mechanism of this recovery, we fused defective PC12 cells with human lymphocytes. A cytogenetic analysis revealed all hybrid clones that recovered neurosecretion to contain a fragment of chromosome 11 including the gene encoding BHC80, a protein of one of the complexes that mediate REST repression. In these clones, REST levels were as high as in defective PC12, whereas BHC80, localized in the nucleus, was 4- to 5-fold higher. Transient transfection of defective PC12 with various amounts of BHC80 cDNA induced (1) in defective PC12, the reexpression of only neurosecretion mRNAs; (2) in defective PC12 cotransfected with the REST negative construct DNA-binding domain (to attenuate gene repression), the recovery of a weak, but complete neurosecretory phenotype, including dense-core granules and their regulated exocytosis. Chromatin immunoprecipitation and immunodepletion analyses revealed the extensive BHC80 association with REST at the genes of two neurosecretion proteins, chromograninB and SNAP25, however only in the low-REST PC12, whereas in high-REST defective PC12 no association was appreciable. In defective PC12 transfected with BHC80 some association was reestablished. Therefore, the recovery of neurosecretion observed after fusion/transfection of defective PC12 depends on the reciprocal level of BHC80 and REST, with BHC80 working as a negative modulator of REST repression. This role appears of possible cell physiological and pathological importance.

The brachial plexus branches to the pectoral muscles in adult rats: morphological aspects and morphometric normative data

Animal models provide an important tool to investigate the pathogenesis of neuromuscular disorders. In the present study, we analyze fiber composition of the brachial plexus branches to the pectoral muscles: the medial anterior thoracic nerve (MATN) and the lateral anterior thoracic nerve (LATN). The morphological and morphometric characteristics and the percentage of motor fibers within each nerve are here reported, adding information to microscopic anatomy knowledge of the rat brachial plexus. As control, we employed the quadriceps nerve, commonly used for the evaluation of motor fibers at hindlimbs. We demonstrated that the MATN and the LATN are predominantly composed of large motor fibers and therefore could be employed to evaluate the peripheral nervous system (PNS) involvement at forelimbs in neurological diseases models, predominantly affecting the motor fiber compartment.

Diffuse intraneural leiomyoma in a case of sensorimotor neuropathy.

Leiomyomas are benign smooth muscle tumors, usually characterized by well-deWned Wbrous pseudocapsule and intersecting fascicles of spindle cells [3]. Few cases of leiomyomas in the peripheral nervous system (PNS) causing external nerve compression have been described [1, 7]. We report the case of a 71-year-old man who was referred for a 2 years history of burning pain and paresthesias associated with slowly progressive weakness of the lower limbs. He underwent total colectomy for diVuse polyposis 2 years before. Neurological examination and nerve conduction studies revealed symmetric sensorimotor axonal neuropathy of the lower limbs. Since all immunological, hematological and cerebrospinal Xuid examinations were negative, a sural nerve biopsy was performed. On semithin plastic sections, a mild degree of endoneurial Wbrosis, reduction of myelinated Wbers and signs of acute axonal degeneration were evident. Some fascicles had greater myelinated Wber loss than others, primarily in the center of the fascicles. Interestingly, paraYn-embedded, hematoxylin and eosinstained sections showed in epineurial and endoneurial perivascular spaces atypical large cells with abundant eosinophilic cytoplasm. Upon electron microscopy, these cells exhibited a basement membrane and prominent pinocytotic vesicles at the periphery (Fig. 1). The cytoplasm was Wlled with actin and myosin Wlaments, aggregates of glycogen particles and few organelles. Some cells showed oval and centrally located nuclei. Immunohistochemically, they were positive for smooth muscle actin, desmin and calponin (Fig. 2), but negative for CD31, cytokeratin and S-100 protein, demonstrating that they were smooth muscle cells (SMC). These cells did not show proliferative activity (negativity for Ki-67). Neither necrosis nor a deWnite capsule were present. The morphological and immunohistochemical data suggested a diagnosis of leiomyoma. This is the Wrst description of a diVuse SMC inWltrate of the PNS, both in the endoneurium and epineurium, whereas well circumscribed and encapsulated leiomyomas have occasionally been reported in the PNS. Other potential tumors such as leiomyosarcoma, schwannoma and hemangioma have to be considered in the diVerential diagnosis, but can be excluded based on immunoproWle [2]. Several hypotheses can be envisaged for the possible cellular origin of this unique tumor in the PNS, including pluripotential mesenchymal cells, perivascular connective tissue elements or SMC of blood vessels [6]. Epineurial proliferation of SMC in sural nerve biopsies indicating inXammatory or non-inXammatory angiopathy has been described before [5]. However, our patient’s sural nerve biopsy revealed SMC also in the endoneurium without signs of angiopathy, suggesting a diVerent origin of SMC. How this inWltrate caused damage of peripheral nerve Wbers remains unclear. Our patient’s clinical presentation suggests a diVuse damage of the PNS, while nerve biopsy Wndings associated with F. Cerri (&) Department of Neurology and INSPE, San RaVaele ScientiWc Institute, Via Olgettina, 6

ZFP423, a transcription factor implicated in Joubert Syndrome and Cerebellar Vermis Hypoplasia, orchestrates the pace and mode of cerebellar neurogenesis

Neurogenesis is a tightly regulated process, both in the embryonic and in the adult brain. Its success depends on the ability of a germinative epithelium to establish the appropriate balance between maintaining an undifferentiated progenitor pool and giving birth to sequential generations of neurons and glia. The Zfp423 gene encodes a 30 Zn-finger transcription factor (TF) which interacts with the SMAD1- SMAD4 complex (BMP signaling), Notch intracellular domain, retinoic acid receptors and Collier/Olf-1/EBF TFs. This gene has been previosly implicated in cerebellar development. Mutations in the human ortholog ZNF423 have been identified in patients carrying cerebellar vermis hypoplasia (CVH) or Joubert Syndrome (JS), and/ or exhibiting other signs of ciliopathy outside the central nervous system. We have been analyzing two mouse mutant lines carrying allelic in-frame deletions of Zfp423. One of them lacks Zn-finger domains 9-20 (Δ9-20), implicated in BMP and Notch signal transduction, while the other lacks a C-terminal domain (Δ28-30). Both mutants exhibit cerebellar malformations and severe ataxia. However, our results indicate that the two protein domains play sharply distinct roles in the context of cerebellar neurogenesis. In Zfp423Δ9-20/Δ9-20 mutants, GABAergic Purkinje cell (PC) neurogenesis is impaired and the PC progenitor pool in the ventricular zone is precociously depleted. Conversely, Zfp423Δ28-30/Δ28-30 mutants display a selective impairment in the development of glutamatergic cerebellar neurons.