Silvia Grifoni

TDP-43 Redistribution is an Early Event in Sporadic Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder consisting of progressive loss of motor neurons. TDP‐43 has been identified as a component of ubiquitin‐immunoreactive inclusions of motor neurons in ALS. We focused on the diffuse cytoplasmic TDP‐43 immunoreactivity in ALS neurons, and quantitatively assessed it in comparison with skein/round TDP‐43 and ubiquitin immunostaining in motor neurons of 30 sporadic ALS cases. The percentage of spinal motor neurons with cytoplasmic TDP‐43 immunoreactivity was higher than that of ubiquitin‐immunoreactive ones. The percentage of TDP‐43‐positive motor neurons was independent of neuron counts in anterior horns, while the percentage of ubiquitinated neurons was inversely correlated. Aiming to define the cytosolic localization of TDP‐43, the immunoblot analysis of spinal cord and frontal cortex showed that full‐length TDP‐43, the 45 kDa form and ubiquitinated TDP‐43 are found in the soluble inclusion‐free fraction. The present data suggest that delocalization, accumulation and ubiquitination of TDP‐43 in the cytoplasm of motor neurons are early dysfunctions in the cascade of the events leading to motor neuron degeneration in ALS, preceding the formation of insoluble inclusion bodies. Being cytoplasmic accumulation an ongoing event during the course of the illness, a therapeutic approach to this incurable disease can be envisaged.

Dementia and cognitive impairment in amyotrophic lateral sclerosis: a review

Amyotrophic lateral sclerosis (ALS) is generally considered to be a paradigm of pure motor neuron disorder; nevertheless, the possible occurrence of cognitive impairment up to a frank dementia in patients affected by ALS is recognized. The appraisal of the cognitive impairment in ALS patients is crucial not only to the therapeutic trials of this incurable disease, but also to the planning of care, compliance to interventions, the end-of-life decisions. The cognitive/behavioral changes of ALS patients are consistent with frontotemporal dysfunctions; the overlap of neuropathological features of ALS and frontotemporal lobe degeneration (FTLD) supports, in addition, the putative spectrum of ALS and FTD. In the present review, the pertinent clinical, genetic, neuropathological, neuropsychological and neuroimaging data of the literature are comprehensively and critically discussed. The distinct and overlapping features of ALS and FTD are pointed out, as well as the undisclosed questions deserving additional studies.

Progranulin expression in brain tissue and cerebrospinal fluid levels in multiple sclerosis

Background: Progranulin (PGRN) is a fundamental neurotrophic factor, and is also involved in inflammation and wound repair. PGRN may have pro- or anti-inflammatory properties, depending upon proteolysis of the anti-inflammatory parent PGRN protein and the generation of pro-inflammatory granulin peptides. Objectives: Our objectives were as follows: (1) to evaluate the presence and distribution of PGRN in multiple sclerosis (MS) brain tissue, correlating it with demyelination and inflammation; (2) to evaluate cerebrospinal fluid (CSF) PGRN concentrations in patients with MS and controls, in relationship to the clinical features of the disease. Methods: Our study involved the following: (1) neuropathological study of PGRN on post-mortem tissue of 19 MS and six control brains; (2) evaluation of PGRN CSF concentration in 40 MS patients, 15 non-inflammatory controls and five inflammatory controls (viral encephalitis). Results: In active demyelinating lesions, PGRN was expressed on macrophages/microglia. In the normal-appearing white matter (NAWM), expression of PGRN was observed on activated microglia. PGRN was expressed by neurons and microglia in cortical lesions and in normal-appearing cortex. No expression of PGRN was observed in controls, except on neurons. PGRN CSF concentrations were significantly higher in patients with relapsing–remitting MS during relapses and in progressive MS patients, compared with relapsing–remitting MS patients during remissions and with non-inflammatory controls. Conclusions: PGRN is strongly expressed in MS brains, by macrophages/microglia in active lesions, and by activated microglia in the NAWM; PGRN CSF concentrations in MS are correspondingly increased in conditions of enhanced macrophage/microglia activation, such as during relapses and in progressive MS.

Cytoplasmic accumulation of TDP-43 in circulating lymphomonocytes of ALS patients with and without TARDBP mutations

TDP-43, encoded by TARDBP, is a ubiquitously expressed, primarily nuclear protein. In recent years, TDP-43 has been identified as the major pathological protein in ALS due to its mislocalisation in the cytoplasm of motor neurons of patients with and without TARDBP mutations and expression in forms that do not match its predicted molecular weight. In this study, the TDP-43 profile was investigated using western immunoblot analysis in whole lysates, nuclei and cytoplasm of circulating lymphomonocytes from 16 ALS patients, 4 with (ALS/TDP+) and 12 without (ALS/TDP−) TARDBP mutations in the protein C-terminal domain, and thirteen age-matched, healthy donors (controls). Three disease-unaffected first-degree relatives of an ALS/TDP+ patient were also included: one carried the parent mutation (Rel/TDP+) whereas the other two did not (Rel/TDP−). In all ALS patients, relatives and controls, TDP-43 retained the predicted molecular weight in whole cell lysates and nuclei, but in the cytoplasm its molecular weight was slightly smaller than expected. In quantitative terms, TDP-43 was expressed at approximately the same levels in whole cell lysates of ALS patients, relatives and controls. In contrast, TDP-43 accumulated in the cytoplasm with concomitant nuclear depletion in all ALS/TDP+ patients, in about 50% of ALS/TDP− patients and in the Rel/TDP+ subject compared to the controls. In the remaining ALS/TDP− patients and in the two Rel/TDP− subjects, TDP-43 matched the control levels in both subcellular compartments. Were these findings further confirmed, circulating lymphomonocytes could be informative of TDP-43 mislocalisation in nervous tissue and used as a biomarker for future disease risk.

Neuropathology of Olfactory Ensheathing Cell Transplantation into the Brain of Two Amyotrophic Lateral Sclerosis (ALS) Patients

Although a large number of amyotrophic lateral sclerosis (ALS) patients have undergone transplantation procedures with olfactory ensheathing cells (OECs) in the Bejing Hospital, to our knowledge, no post‐mortem neuropathologic analyses have been performed. We examined the post‐mortem brain of two Italian patients affected by ALS who underwent cellular transplantation in Beijing with their consent. Our aim was to assess the events following the graft procedure to possibly support the rationale of the treatment strategy. The neuropathologic findings were analyzed on the basis of the limited awareness of the experimental conditions and discussed in relation to the safety, efficacy and long‐term outcome of the transplanted cells. Islands of quiescent, undifferentiated cells within the delivery track persisting for up to 12 months–24 months were found. Prominent glial and inflammatory reaction around the delivery track strongly supports the encasement of the graft. Evidence of axonal regeneration, neuronal differentiation and myelination was not seen. The surgical procedure of implantation was not compatible with a neurotrophic effect. The OEC transplantation did not modify the neuropathology of ALS in the two patients. In conclusion, the present neuropathologic analysis does not support a beneficial effect of fetal OEC implantation into the frontal lobes of ALS patients.

Alterations of myelin‐specific proteins and sphingolipids characterize the brains of acid sphingomyelinase‐deficient mice, an animal model of Niemann–Pick disease type A

Niemann–Pick disease (NPD) type A is a neurodegenerative disorder caused by sphingomyelin (SM) accumulation in lysosomes relying on reduced or absent acid sphingomyelinase (ASM) activity. NPD‐A patients develop progressive neurodegeneration including cerebral and cerebellar atrophy, relevant Purkinje cell and myelin deficiency with death within 3 years. ASM‘knock‐out’ (ASMKO) mice, an animal model of NPD‐A, develop a phenotype largely mimicking that of NPD‐A. The mechanisms underlying myelin formation are poorly documented in ASMKO mice. In this study we determined the content of four myelin‐specific proteins, myelin basic protein (MBP), 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP), myelin associated glycoprotein (MAG) and proteolipid protein (PLP), and that of myelin‐enriched sphingolipids in the brains of ASMKO and wild‐type mice in early stages of post‐natal (pn) life. Protein and mRNA analysis revealed that in ASMKO mice beginning from 4 post‐natal weeks (wk‐pn), the expression levels of MAG, CNP, and MBP were below those observed in wild‐type mice and the same applied to PLP at 10 wk‐pn. Moreover, at 4 wk‐pn the expression of SOX10, one of the transcription factors involved in oligodendrocyte development and maintenance was lower in ASMKO mice. Lipid analysis showed that SM and the gangliosides GM3 and GM2 accumulated in the brains of ASMKO mice, as opposed to galactocerebroside and galactosulfocerebroside that, in parallel with the mRNAs of UDP‐galactose ceramide galactosyltransferase and galactose‐3‐O‐sulfotransferase 1, the two transferases involved in their synthesis, decreased. Myelin lipid analysis showed a progressive sphingomyelin accumulation in ASMKO mice; noteworthy, of the two sphingomyelin species known to be resolved by TLC, only that with the lower Rf accumulated. The immunohistochemical analysis showed that the reduced expression of myelin specific proteins in ASMKO mice at 10 wk‐pn was not restricted to the Purkinje layer of the cerebellar cortex but involved the cerebral cortex as well. In conclusion, reduced oligodendrocyte metabolic activity is likely to be the chief cause of myelin deficiency in ASMKO mice, thus shedding light on the molecular dysfunctions underlying neurodegeneration in NPD‐A.

Absence of TARDBP Gene Mutations in an Italian Series of Patients with Frontotemporal Lobar Degeneration

Background/Aim: Recent studies showed that TAR DNA-binding protein 43 (TDP-43), encoded by the TARDBP gene, is a major pathological protein in both sporadic and familial frontotemporal lobar degeneration (FTLD). The aim of this study was to search for mutations of the TARDBP gene in the disease. Methods: We sequenced the TARDBP gene in 172 unrelated FTLD patients recruited from 2 Italian memory clinics. Results: We identified 3 different variants of the TARDBP gene in 12 FTLD patients. Three patients showed a silent variant, Ala66Ala (c.332T → C) in exon 2. A novel heterozygous mutation was found in intron 4 (c.543 + 51A → G) in 1 patient, which is not located at the splicing site. Finally, a c.208C → T variant in the 3′ untranslated region was detected in 8 probands. None of the aforementioned variants were predicted to affect TDP-43. Hence, pathogenic mutations were not identified in any of the FTLD cases. Conclusion: Our study, in accord with previous studies in different populations, found no evidence for a major genetic role of the TARDBP gene in FTLD.

Heterozygous individuals with mild phenotype in late-onset glycogen storage disease type 2: a new cohort of patients?

Late-onset glycogen storage disease type 2 (GSD2) is a genetic but heterogeneous disorder, which may present anywhere along a continuum of severity from an isolated hyperCKemia to a profound, generalized muscle weakness with pulmonary involvement. The gold standard for diagnosis is confirmation of low or absent levels of acid alpha-glucosidase (GAA) enzyme activity (usually in the range of 1-40% of normal levels), which is confirmed only in some cases by molecular analysis of the GAA gene. In the literature, heterozygous individuals are usually considered to be asymptomatic, although they can have reduced enzymatic activity. Since enzyme replacement therapy (ERT) became available in 2006, it has improved the prognosis for severe infantile-onset Pompe disease, as well as for late-onset forms by improving muscle/respiratory function and/or stabilizing clinical progression. Because the disease is now treatable, it is essential to understand which patients may benefit from ERT.

N-CAM Dysfunction and Unexpected Accumulation of PSA-NCAM in Brain of Adult-Onset Autosomal-Dominant Leukodystrophy

Previously, myelin from cerebral white matter (CWM) of two subjects of a family with orthochromatic adult‐onset autosomal‐dominant leukodystrophy (ADLD) was disclosed to exhibit defective large isoform of myelin‐associated glycoprotein (L‐MAG) and patchy distribution only in the elder subject. L‐MAG and neural cell adhesion molecule (N‐CAM) (N‐CAM 180, 140, and 120) are structurally related and concur to myelin/axon interaction. In early developmental stages, in neurons and glia N‐CAM is converted into polysialylated (PSA)‐NCAM by two sialyltransferases sialyltransferase‐X (STX) and polysialyltransferase‐1 (PST). Notably, PSA‐NCAM disrupts N‐CAM adhesive properties and is nearly absent in the adult brain.